Introduction

Hoppea fastigiata (Griseb.) C.B. Clarke (Gentianaceae) is a xanthone-producing annual medicinal plant restricted to only a few ecological niches in India. Xanthones are a class of phenolic compounds (secondary metabolites) restricted to only a few higher plant families such as Gentianaceae and Clusiaceae. Nevertheless, xanthones represent a class of natural products with interesting pharmacological properties [1]. Several xanthones from whole parts of H. fastigiata have been isolated and elucidated through chemical and spectroscopic analyses. These include 1,7-dihydroxy-3,5-dimethoxyxanthone [2], 1,5-dihydroxy-3,7-dimethoxyxanthone [3], 1,5,7-trihydroxy-3-methoxyxanthone [4], 1,3,8-trihydroxy-7-methoxyxanthone [5] and 1,3,6-trihydroxy-8-methoxyxanthone [6]. Xanthones isolated from several Gentianaceae members in temperate regions showed broad similarity with the chemical structures reported from H. fastigiata; these xanthones were also shown to be potent inhibitors of the enzymes acetylcholinesterase (AChE) and monoamine oxidase (MAO) [7].

AChE is linked with Alzheimer’s disease (AD), a CNS-related disorder mostly prevalent in older populations. It is responsible for impaired cognitive function and progressive memory loss [8, 9]. Due to loss of cholinergic neurons, there is a deficit of synaptic acetylcholine. In addition, the enzyme AChE degrades the neurotransmitter acetylcholine, and has been shown to accelerate AD [10]. The availability of acetylcholine can be increased by inhibiting AChE, which is responsible for the hydrolysis of acetylcholine [11]. MAO is involved in the metabolism of neurotransmitters such as dopamine, adrenaline and noradrenaline and in the inactivation of exogenous arylalkyl amines [12]. There are two sub-forms of the MAO enzyme, MAO-A and MAO-B. MAO-A is linked with depression, which is caused by the deregulation and depletion of monoamine neurotransmitters because of upregulation of MAO-A activity, and MAO-B is linked with Parkinson’s disease (PD) which is caused by a loss of neurons from the substantia nigra of the brain which stops producing dopamine and affects the brain’s ability to control movement [13]. MAO-A and B also play an important role as a first line of defence against the food-derived monoamines such as tyramine and 3-phenylethanolamine, absorption of which causes sympathomimetic effects such as a rise in blood pressure, which is often known as the “cheese effect” [14].

One of the plant-based drugs approved by US Food and Drug Administration (FDA) used in the treatment of AD is physostigmine, which serves as an excellent inhibitor of AChE but suffers from poor intestinal absorption in humans [15]. Likewise, the traditional irreversible MAO inhibitors show lack of selectivity for MAO sub-forms, which may lead to the cheese effect. Thus, there is increasing interest in finding better AChE and MAO inhibitors showing good brain penetration and high bioavailability and reversible selectivity [14]. Xanthone compounds isolated from Gentiana campestris, Gentianella amarella and Garcinia mangostana showed potent inhibitions against in vitro AChE and MAO activities [7, 16, 17].

H. fastigiata was known for its uses by several tribal peoples in various brain-related ailments. Use of this plant in the Indian systems of medicine for the treatment of haemorrhoids, cardiac dropsy and in certain mental disorders including epilepsy was also known [18]. It was therefore necessary to evaluate the extracts of H. fastigiata for any AChE and MAO inhibitory properties. Recently, crude extracts of wild H. fastigiata were shown to possess in vitro antioxidant capacities and acetylcholinesterase (AChE) inhibition activity [19]. However, no information is available on the chemical compounds of H. fastigiata that were responsible for such biological activities.

One of the major problems with the collection of H. fastigiata biomass in the field is its seasonal nature, and its life-cycle duration of only 6–8 weeks. Moreover, this tiny hygrophytic plant is very restricted in distribution and is consequently included in the IUCN Red List of Threatened Species. Therefore, to explore the full bioprospecting of H. fastigiata, ex situ multiplication through in vitro propagation appears to be an attractive strategy for sustainable utilization of this fragile bioresource. As xanthones were reported mostly from aerial parts of H. fastigiata, we anticipated that establishment of shoot cultures could be a suitable platform for production of xanthones [4]. This raison d’être is based on the expression of pathway enzymes between disorganized and organized cultures, as elucidated with Mitragyna speciosa shoot cultures [20].

This study aimed to establish for the first time in vitro shoot cultures of H. fastigiata, which could serve as a sustainable source for this species for effective utilization. The major xanthones produced in shoot cultures of H. fastigiata were isolated and identified. The efficacies of the isolated xanthones were evaluated for their antioxidant capacities and AChE or MAO inhibitory properties. We also report the inhibition-kinetic characteristics of isolated xanthones against eel AChE and human recombinant MAO-A and MAO-B enzymes.

Materials and methods

Chemicals

AChE from Electrophorus electricus (eel), human recombinant MAO-A and MAO-B, acetylthiocholine iodide (ATChI), 5,5-dithiobis-2-nitrobenzoic acid (DTNB), kynuramine dihydrobromide and 4-quinolinol were purchased from Sigma-Aldrich India Pvt Ltd. Naphthyl acetate, fast blue B salt, 1-naphthalene acetic acid (NAA), 6-benzylaminopurine (BA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), potassium persulfate, 2,4,6-tripyridyl-s-triazine (TPTZ) and Folin–Ciocalteu reagent were purchased from Himedia Laboratories Pvt Ltd, India. All the organic solvents were of analytical grade and were purchased from SRL, India.

In vitro propagation of H. fastigiata

Nature-grown plants were collected from the Bolpur forest (Santiniketan), West Bengal, India. The plant was identified on the basis of reproductive morphology and by the Department of Botany, Visva-Bharati University, and a voucher specimen collection number VBH-129-89 was deposited in the herbarium. H. fastigiata explants (apical meristem, stem, leaf and root) were washed thoroughly and kept in running water for 30 min. They were then treated with 0.1 % of Triton-X100 for 5–10 min. Afterwards, these explants were washed with double-distilled water for 5–10 min. Subsequently, explants were immersed in 70 % (v/v) ethanol for 2–3 min and washed 2–3 times with double-distilled water. Eventually, the explants were treated with an aqueous solution of 0.1 % HgCl2 and rinsed 2–3 times with sterile water to remove all traces of HgCl2. The sterilized explants were then inoculated on Murashige and Skoog (MS) medium with different concentrations and combinations of hormones such as NAA, IAA and BA. The growth index was calculated according to the following equation:

$${\text{Growth}}\;{\text{index}} = {\text{Final}}\;{\text{FW}}\;{\text{of}}\;{\text{tissue}} - {\text{Initial}}\;{\text{FW}}\;{\text{of}}\;{\text{tissue}}/{\text{initial}}\;{\text{FW}}\;{\text{of}}\;{\text{tissue}}$$

where FW = fresh weight.

Extraction, isolation and characterization of compounds

Extracts were prepared using four different solvents, namely water, ethanol, ethyl acetate and petroleum ether. In vitro shoots (0.25 g) were dipped in the solvents (15 ml each) overnight and crushed using a mortar and pestle. The resulting extract was then subjected to ultrasonic extraction for 30 min. It then was centrifuged at 8000g for 10 min (MiniSpinplus, Eppendorf). After drying the supernatant either in a vacuum rotary evaporator or vacuum concentrator (Concentrator plus, Eppendorf) as appropriate, the final extraction yields (%) were calculated using the formula [W 2 − W 1/W 0], where W 0, W 1 and W 2 are the weights of initial plant material, container without extract and container with extract, respectively.

The dried ethanolic extract was re-suspended in 1 ml ethanol, filtered through a 0.45 syringe filter and analyzed by HPLC (Waters) for separation and subsequent isolation of compounds. A Symmetry™ (Waters) C18 reverse-phase column (3.5 μm, 75 × 4.6 mm) was used and the detection of compounds was simultaneously monitored at 254 and 365 nm. The mobile phase composition of solvents A and B were 1 mM trifluoroacetic acid (TFA) in deionized water and methanol (MeOH), respectively. At time 0 min, A and B were 90 and 10 %, respectively. After 60 min, the percentages of A and B were 0:100 with a flow rate of 1 ml/min. The compounds eluted at the retention times of 30 min (compound 1), 36 min (compound 2) and 52 min (compound 3) were subsequently collected. LC–MS analysis of these compounds was carried out on a QTrap 3200 system (Applied Biosystems/MDS Sciex) at Technische Universität Braunschweig as described in the literature [21]. All NMR data were collected on a Bruker Advance AMX-500 MHz FT-NMR spectrometer located at NMR Facility, Indian Institute of Technology Roorkee.

Comparative analysis of major xanthones from nature-grown and in vitro shoot cultures of H. fastigiata

Comparative analysis was performed by three different samples of nature-grown (7 and 30 days after germination and completely mature plant) and shoot cultures (after 7, 15 and 30 days of sub-culture) of H. fastigiata. The extraction was made in ethanol and the supernatant was used for HPLC analysis. The data are represented as average of compounds at different time intervals.

DPPH free radical scavenging assay

DPPH assay was performed according to a previously described method [22]. The free radical scavenging activity of the extracts was tested using DPPH free radical. Crude extracts of plant (0.1 ml) were mixed with the 0.9 ml ethanolic solution of DPPH (0.1 M). The mixture was vortexed and kept in the dark for 30 min at room temperature. The decrease in optical density was monitored at 517 nm.

ABTS free radical scavenging assay

ABTS assay was performed according to a previously described method with slight modifications [23]. ABTS was dissolved in water to 7 mM concentration. The ABTS free radical (ABTS·) was prepared by adding 2.45 mM potassium persulfate to ABTS solution and incubating in the dark for 12–16 h. For the working solution, the absorbance of the ABTS solution was adjusted by diluting it with ethanol to optical density 0.7 (±0.02) at 734 nm. The reaction mixture consisted of working solution (995 µl) and crude extract (5 µl). The mixture was then vortexed and incubated for 6 min at room temperature and the decrease in absorbance was monitored at 734 nm.

FRAP assay

The reducing power of the extract was determined by ferric reducing antioxidant power (FRAP) assay [24] with slight modifications. Stock solutions comprised TPTZ (2,4,6-tripyridyl-s-triazine) solution (10 mM) which was prepared in HCl (40 mM), FeCl3·6H2O (20 mM) and acetate buffer (300 mM, pH 3.6). The fresh working solution was prepared by mixing 25 ml acetate buffer, 5 ml TPTZ and 5 ml FeCl3·6H2O. The extracts (3 µl) were allowed to react with FRAP working solution (997 µl). The mixture was then incubated for 6 min at 37 °C and absorbance was recorded at 593 nm.

Determination of total phenolic content

Estimation of total phenolic content (TPC) was done as described previously [25]. Crude extract of plant (100 µl) was mixed with 200 µl of 10 % Folin–Ciocalteu reagent and 800 µl of Na2CO3 (700 mM) and vortexed vigorously. The tubes were incubated for 2 h at room temperature. Increase in absorbance was then measured at 765 nm. TPC value was obtained by comparing the change in absorbance of the solution containing the extract at 765 nm with that of a calibration plot of gallic acid and was expressed as mg gallic acid equivalent/g fresh mass.

Acetylcholinesterase (AChE) inhibitory activity

The AChE inhibitory property of different solvent extracts was studied as described previously with slight modifications. The reaction mixture consisted of 500 μl of 3 mM DTNB, 46 μl of 15 mM ATChI, 356 μl of 50 mM Tris–HCl buffer (pH 8.0), sample extract and 100 μl of 0.28 U/ml AChE. The reaction mixture was monitored at 405 nm for 5 min. For IC50 value calculations, the absorbance was recorded after incubating reaction mixtures for 10 min. The rate of reaction before addition of the enzyme was subtracted from the reaction rate after addition of the enzyme in order to nullify the absorbance changes due to spontaneous hydrolysis of the substrate. Percentage inhibition was calculated by comparing the rate of reaction for the sample with that for the negative control [26]. To study the inhibition kinetics of the compounds, the reaction mixtures were continuously monitored at various substrate (ATChI) concentrations ranging from 100 to 700 µM and two inhibitor concentrations of 360 and 500 µM.

TLC bioautography

The AChE inhibitory activity of individual compounds was tested by the TLC bioautography method [27] with slight modifications. Samples were allowed to run onto silica gel 60 F254 plates which were later sprayed with the enzyme stock solution and incubated at 37 °C for 20 min. After incubation the plates were sprayed with 10 ml of naphthyl acetate (2.5 mg/ml) and 40 ml Fast Blue B salt (2.5 mg/ml). The plates were then dried and observed for white spots in comparison to the purple background. Naphthyl acetate and AChE enzyme were mixed prior to spraying onto the TLC plate to eliminate false-positive results.

Monoamine oxidase inhibition assay

MAO-A and MAO-B inhibitions were studied using recombinant human MAO enzymes as described previously [28] with slight modifications. For MAO-A inhibition, 1 ml of reaction mixture consisted of 950 μl of 0.1 M phosphate buffer containing 20.2 mM KCl, 0.7 U enzyme and 45 μl of 2 mM kynuramine dihydrobromide. For MAO-B, all reaction mixture components were the same except that 30 μl of 2 mM kynuramine was added. The reaction product was monitored at 329 nm for 5 min. Percentage inhibition was calculated by comparing the rate of reaction for the sample to that for the negative control. Finally, the reaction mixture was analyzed by HPLC to confirm the product formation by comparing their retention times with the standard 4-quinolinol obtained commercially (Sigma-Aldrich). HPLC was monitored at 329 nm at a gradient mode of 10–100 % B for 60 min and then isocratic elution at 100 % B with a flow rate of 1 ml/min. The mobile phase composition of solvents A and B was 1 mM TFA in deionized water and MeOH, respectively. To study the inhibition kinetics of the compounds, the reaction mixtures were continuously monitored at various substrate (kynuramine) concentrations ranging from 10 to 200 µM and two inhibitor concentrations of 12 and 14 µM.

Statistical analyses

All data are expressed as mean ± standard error (SE). The statistical analysis was performed using the ‘Multi-Variate Statistical Package’ (MVSP version 3.22). Principal component analysis (PCA) was performed to evaluate relationships between the different extraction solvents (ethanol, aqueous, ethyl acetate and petroleum ether) and the different analytical methods (DPPH, ABTS, FRAP and TPC for antioxidant capacities and AChE, MAO-A and MAO-B inhibitory activities for neuroprotective properties). Two principal components with the greatest eigenvalues were selected. Extraction solvents served as dependent variables whereas DPPH, ABTS, FRAP, TPC, AChE, MAO-A and MAO-B activities served as explanatory variables. Results are shown as the means of values and SE of independent measurements (n = 3) and a result was considered statistically significant when the p value was <0.05. The means were compared using one-way ANOVA followed by Duncan’s multiple range tests.

Results and discussion

In vitro propagation of H. fastigiata

In vitro cultures of H. fastigiata were established by using different combinations and concentrations of plant growth regulators in MS basal medium. Different phytohormones such as BA, kinetin, NAA, IAA and IBA were supplemented in the media either singly or in combination. Apical meristem, when used as explant, showed 100 % response in direct regeneration of shoots on the medium containing cytokinin (either BA or NAA), which initiates cell division and differentiation. Induced shoots were separated from explants and sub-cultured in the medium containing IBA (1 mg/L) which resulted in the formation of roots after 35 days of sub-culture (Supplementary Material). Subsequently, the plantlets were maintained in the medium containing a range of combinations of plant growth regulators: NAA (0.5 mg/L) and BA (0.5 mg/L), NAA (1 mg/L) and BA (0.2 mg/L), BA (0.5 mg/L) and kinetin (0.5 mg/L), BA (1 mg/L) and kinetin (0.1 mg/L), and BA (4 mg/L) and IAA (1 mg/L) which showed a promising outcome. BA (1 mg/L) and kinetin (0.1 mg/L) combinations were found to be the most efficacious of all the combinations. It was earlier reported that a positive role of synergistic effects of BA and kinetin in regulation of ethylene synthesis resulted in a high frequency of shoot organogenesis [29]. Thus it appears that BA (1 mg/L) and kinetin (0.1 mg/L) proved effective in maintaining the ethylene levels of the plantlets and faster growth of H. fastigiata. Using this combination, a growth index of 0.9, 15 ± 2 shoots per explant and height of 2.7 ± 0.3 cm was achieved after 30 days. The explants were incubated at 25 °C under a 16-h light photoperiod. This was the highest biomass growth achieved using BA (1 mg/L) and kinetin (0.1 mg/L), as compared to other combinations of plant growth regulators (Table 1; Supplementary Material). The cultures were sub-cultured after every 60 days, when the highest total biomass yield (25 ± 3 mg fresh weight) was recorded in the MS medium containing BA (1 mg/L) and kinetin (0.1 mg/L). A positive correlation between ethylene synthesis and tissue browning under in vitro conditions was also reported earlier [30]. Of all the selected combinations of plant growth regulators, BA (1 mg/L) and kinetin (0.1 mg/L) showed a decrease in tissue browning and subsequent enhancement of regeneration and cell differentiation of H. fastigiata.

Table 1 Influence of different combinations of phytohormones on growth index, number of shoots and height of H. fastigiata
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Isolation and identification of compounds

The final extraction yields for aqueous, ethanol, ethyl acetate and petroleum ether solvent extracts were 15.13, 11.21, 6.92 and 6.25 %, respectively. HPLC analysis of ethanolic extract showed the presence of three major compounds (Fig. 1). Final extraction yields for compounds 1, 2 and 3 after HPLC purification were 1.25, 0.63 and 0.59 %, respectively. Compounds 1 and 2 were structurally identified as 1,5,7-trihydroxy-3-methoxyxanthone and 1,5-dihydroxy-3,7-dimethoxyxanthone, respectively, by comparing with the standards (available at Braunschweig), and also using the ESI–MS and UV–Vis spectral information available in the literature [3, 4]. Compound 3: yellow crystalline, m.p. 240–242 °C; ESI–MS m/z [M + H]+ 273.1, 257.9, 230, 215.1, 201.1, 187; UV λ max (CH3OH) nm 247, 310; (CH3OH + AlCl3) nm 247, 324; (CH3OH + HCl) nm 247, 310, (CH3OH + NaOAc) nm 247, 315; 1H-NMR (500 MHz, CD3OD): δ 3.91 (3H, s, C8-OCH3), 6.33 (1H, d, J = 8.5 Hz, H-4), 6.61 (1H, d, J = 8.5 Hz, H-2), 6.9 (1H, d, J = 8.5 Hz, H-6), 7.6 (1H, d, J = 8 Hz, H-7). On the basis of TOCSY, HSQC and ROESY spectra the structural identity of the compound was confirmed as 1,3,5-trihydroxy-8-methoxyxanthone (Supplementary Material).

Fig. 1
figure 1

Representative HPLC chromatogram showing detection of three compounds from ethanolic extract of H. fastigiata shoot culture. These three compounds were subsequently identified as 1,5,7-trihydroxy-3-methoxyxanthone (1), 1,5-dihydroxy-3,7-dimethoxyxanthone (2) and 1,3,5-trihydroxy-8-methoxyxanthone (3) on the basis of their UV–Vis spectral properties, mass spectroscopy and NMR analyses

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Comparative analysis of three xanthones from nature-grown and in vitro shoot cultures of H. fastigiata

Comparative analysis of shoots from nature-grown plants and in vitro cultures showed that the content of 1 was 5.3-fold higher in shoot cultures than in wild plants; the content of 2 was 25-fold higher in shoot cultures. The content of 3 was 1.4-fold higher in the wild plants than the shoot cultures (Fig. 2). It was interesting to note that H. fastigiata in vitro cultures, when grown in BA (1 mg/L) and kinetin (0.1 mg/L) medium, produced much higher contents of 1 and 2 than nature-grown plants. Cytokinins are known to enhance the production of secondary metabolites and to play an important role in cytodifferentiation and sub-cellular differentiation, such as production of anthocyanins in Camptotheca acuminata [31, 32]. Cytokinin and auxin are also known to modulate secondary metabolites production in vitro, as reported in Commiphora wightii and Pueraria tuberosa for terpenoids and isoflavonoids synthesis, respectively [33]. The content of 3 was higher in nature-grown plants. This could be explained by the fact that under field conditions plants are exposed to various abiotic stresses such as salinity, temperature, etc. which result in the production of certain secondary metabolites, since abiotic stresses are known to affect secondary metabolism [34].

Fig. 2
figure 2

Box-and-whiskers plots showing comparative analysis of xanthone contents in wild and shoot cultures of H. fastigiata. a 1,5,7-trihydroxy-3-methoxyxanthone (1) content, b 1,5-dihydroxy-3,7-dimethoxyxanthone (2) content, c 1,3,5-trihydroxy-8-methoxyxanthone (3) content

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AChE inhibitory activity from extracts of shoot culture

Extracts were prepared from in vitro cultures in four different solvents (aqueous, ethanol, ethyl acetate and petroleum ether). Testing of AChE inhibitory activity for all the different extracts was carried out followed by subsequent determination of IC50 values. It was observed that the ethanolic extract was most active, with the lowest IC50 value (157 ± 13 µg/ml), which was 97.06, 92.5 and 87.5 % lower than the aqueous, ethyl acetate and petroleum ether extracts, respectively (Table 2). Thus, the most active ethanolic fraction was subsequently tested for the presence of the active principle of AChE inhibition. Ethanolic extract of H. fastigiata was screened by TLC bioautography (Supplementary Material). Naphthyl acetate when used as substrate binds to the AChE enzyme and gives a purple-coloured complex. Upon binding with an inhibitor, the enzyme hinders enzyme–substrate interaction causing inactivation of the AChE enzyme, which fails to produce a coloured complex. Thus, the regions of the TLC plate which contain AChE inhibitors show up as white/discolored spots against the purple background, thus indicating the presence of AChE inhibitor(s) [27]. Based on TLC bioautography results, we identified three compounds as apparent inhibitors of the AChE enzyme. The AChE inhibitory property of compounds was confirmed by a colorimetric method [26]. Of the three compounds, 3 was most potent inhibitor of AChE with an IC50 value of 97 ± 11 µg/ml.

Table 2 IC50 values for AChE, MAO-A and MAO-B by different solvents and isolated compounds of H. fastigiata
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MAO-A and MAO-B inhibitory activity from extracts of shoot culture

Extracts were prepared from in vitro cultures in four different solvents (aqueous, ethanol, ethyl acetate, and petroleum ether). It was observed that of all the extracts the ethanolic extract was the most active for MAO-A (IC50 value 25 ± 4 µg/ml) and MAO-B (IC50 value 68 ± 18 µg/ml) when compared to the aqueous, ethyl acetate and petroleum ether extracts (Table 2). The isolated compounds were tested for their selective capability to inhibit MAO-A or MAO-B. Compounds 1 and 2 showed inhibition of MAO-A, while compounds 2 and 3 showed inhibition of MAO-B. Compounds 1 and 3 showed selective inhibition of MAO-A and MAO-B, respectively. This could provide a good opportunity for the utilization of these compounds for selective treatment of depression or PD. The reaction was confirmed by HPLC where the formation of reaction product (4-quinolinol) was monitored at 329 nm. The HPLC profile showed less 4-quinolinol formation in the MAO-A reaction than the MAO-B reaction containing ethanolic extract (Fig. 3). These results are supported by the spectrophotometric assay where the IC50 value for ethanolic extract against MAO-A was lower than MAO-B, as indicated earlier. Because of comparable inhibition properties of these isolated compounds with that of selective standards, inhibition kinetics studies were performed to determine the type of inhibition.

Fig. 3
figure 3

Representative HPLC chromatograms for monoamine oxidase assay. a Standards [*4-quinolinol (product)]; b reaction terminated at 0 min; c control reaction with no inhibitor; d reaction showing the inhibition of MAO-A enzyme by ethanolic extract; e reaction showing the inhibition of MAO-B enzyme by ethanolic extract. All the reactions were performed for 60 min, except for reaction B which was terminated at 0 min

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Evaluation of inhibition mechanism and kinetics of AChE, MAO-A and MAO-B

Detailed in vitro studies were carried out to understand the kinetics and mechanism of inhibition of eel AChE, recombinant human MAO-A and MAO-B by isolated compounds. Inhibitors were tested at varying concentrations depending on their IC50 values against AChE, MAO-A and MAO-B.

For AChE inhibition, varying concentrations of ATChI were used to study the nature of inhibition of this enzyme. The enzyme kinetics was plotted as a Lineweaver–Burk plot (Fig. 4). From the Michaelis–Menten equation based plotting, K m, V max, K cat, K cat/K m and K i values were calculated for the activity of control and compounds. For compound 1, it was observed that the K m value increased and V max value decreased compared to control. This suggested the probability of mixed type of inhibition of AChE. For compound 2, the K m value of the enzyme with and without inhibitor remained the same (100 µM) whereas the V max value decreased, which indicated a non-competitive inhibition of AChE. It has been observed that substitution of hydroxyl, methoxyl, allyloxy and prenylated oxyl at the C-3 position in the xanthone skeleton with a methoxyl group at the C-3 position was the most effective in inhibiting AChE [35]. This supported the inhibitory properties of both compounds 1 and 2 which possessed a methoxyl group at the C-3 position. For compound 3, the K m value increased and V max value decreased, which also indicated the mixed type of inhibition. The K cat value indicated the turn-over number of the AChE enzyme; all three compounds reduced the turn-over number but 3 was most effective. Compound 3 was most effective in reducing the catalytic efficiency (K cat/K m) of the enzyme followed by 1 and 2, respectively (Table 3). Inhibition constant (K i) values were also calculated for all three xanthones. When the inhibitor binds with the enzyme it is denoted as K ia and when the inhibitor binds with the enzyme–substrate complex it is denoted as K ib.

Fig. 4
figure 4

Lineweaver–Burk plots for AChE dependence on substrate concentration (100–700 µM). Kinetics characteristics of inhibition of eel AChE with a 1,5,7-trihydroxy-3-methoxyxanthone (1), b 1,5-dihydroxy-3,7-dimethoxyxanthone (2), c 1,3,5-trihydroxy-8-methoxyxanthone (3). V = µmol/min and S = substrate acetylthiocholine iodide (ATChI) concentration (µM)

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Table 3 Kinetic parameters of AChE, MAO-A and MAO-B (with and without inhibitor)
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For MAO-A, a nonselective substrate kynuramine was used at varying concentrations to determine the type of inhibition. Based on the IC50 values, the inhibitor concentration was fixed for the three compounds. The kinetics data plotted as a double reciprocal Lineweaver–Burk plot (Fig. 5) suggested that compound 1 showed a decrease in V max value along with the decrement of K m value, which suggested the mixed reversible type of inhibition. Compounds bearing an –OH group at C-1 and –OCH3 at C-3 positions showed more activity against the MAO-A enzyme [36]. Compound 2 showed the same V max value with the increment of K m value depending on the dose of inhibitor, which suggested the competitive reversible type of inhibition. For MAO-B also, kynuramine was used at varying concentrations and the data were plotted as a Lineweaver–Burk plot. The plot suggested that compound 2 showed the same V max value with the increment of K m value, which indicated the competitive reversible type of inhibition. Compound 3 showed a decrement in V max value and increment in K m value, indicative of the mixed type of inhibition. Compounds 2 and 3 were most effective in reducing the catalytic efficiency (K cat/K m) of MAO-A and MAO-B, respectively.

Fig. 5
figure 5

Lineweaver–Burk plots for MAO-A and MAO-B dependence on substrate concentration (10–200 µM). Kinetics characteristics of inhibition of human recombinant MAO-A with a 1,5,7-trihydroxy-3-methoxyxanthone (1), b 1,5-dihydroxy-3,7-dimethoxyxanthone (2); kinetics characteristics of inhibition of human recombinant MAO-B with c 1,5-dihydroxy-3,7-dimethoxyxanthone (2), d 1,3,5-trihydroxy-8-methoxyxanthone (3). V = µmol/min and S = substrate kynuramine concentration (µM)

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Antioxidant capacities of crude extracts and compounds isolated from H. fastigiata shoot cultures

Antioxidant activities of H. fastigiata were also studied in the crude extracts of four different solvents (ethanol, aqueous, ethyl acetate and petroleum ether). The TPC of the ethanolic extract was found to be highest, followed by ethyl acetate, aqueous and petroleum ether extracts which showed the lowest values, indicating that the ethanol was most suitable solvent. DPPH activity showed the lowest IC50 value in the ethanolic extract, followed by ethyl acetate, aqueous and petroleum ether, thus indicated that ethanolic crude extract was most effective in scavenging the DPPH free radical. The antioxidants which are capable of donating protons to free DPPH radicals fade the violet colour which ultimately turns colorless depending upon the strength of antioxidants. Similarly, the ABTS assay was also done which showed that the ethanolic extract had a higher ABTS value, followed by ethyl acetate, aqueous and petroleum ether extracts. The ABTS free radical (ABTS·) is teal blue in colour. The antioxidants which are effective in neutralizing the free radicals by donating the lone pair of electrons to ABTS· are capable of decolorizing the ABTS solution. Thus, the less blue colour developed after reaction, the higher the potency of antioxidants to donate the free electrons. FRAP values also showed a similar trend, in which the ethanolic extract was most potent antioxidant, followed by ethyl acetate, aqueous and petroleum ether. All data suggested that the ethanolic extract has the best capacity to scavenge the free radicals. It was earlier shown that the ethanolic solvent was best in terms of extraction and antioxidant capacities in nature-grown plants. TPC in nature-grown plants was reported as 134.4 ± 3.1 mg GAE g−1 dry weight [19], which was very high compared to in vitro cultures. This exceptionally high level of TPC was perhaps the result of various stress factors under field conditions. Thus, a positive correlation between the high TPC and antioxidant activities was responsible for the high antioxidant capacities of ethanolic extracts [37]. The three compounds were also tested for DPPH, ABTS and FRAP assay; compound 2 (DPPH IC50 value = 0.0833 ± 0.003 mg/ml) showed strong antioxidant capacity followed by compound 1 (DPPH IC50 value = 0.4092 ± 0.002 mg/ml) and compound 3 (DPPH IC50 value = 0.902 ± 0.001 mg/ml) (Table 4). The antioxidant capacities of the three xanthones identified from ethanolic extract also support the strong activity of the ethanolic extract. The DPPH IC50 of ascorbic acid is 0.00207 mg/ml and two xanthones, cudraxanthone and macluroxanthone, isolated from Cudrania tricuspidata showed DPPH IC50 values of 10.4 and 15.4 µM, respectively [38].

Table 4 Antioxidant activities of crude extracts and compounds isolated from H. fastigiata
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Statistical analyses

Statistical analysis was done using PCA. Four different extracts were tested to detect the most suitable solvent for extraction of in vitro shoot cultures of H. fastigiata for antioxidant capacities (DPPH, ABTS, FRAP, TPC) and neuroprotective capacities (AChE, MAO-A and MAO-B inhibition). From the graphical representation (scatter-plot) of PCA it was clear that the first (PC 1) and second (PC 2) principal components were sufficient to explain the total variance, i.e. 99.98 % for antioxidant capacities. Three completely different groups could be clearly distinguished from the graph. Group I was located on the left side of the scatter-plot, and consisted of ethanol and ethyl acetate. Group II and group III included aqueous and petroleum ether, respectively. Ethanol and ethyl acetate extract can be considered to have similar potencies in free-radical scavenging capacity. Both members of group I possessed a higher amount of TPC along with higher antioxidant capacity. Similarly, for neuroprotective capacities, it was clear that the first (PC 1) and second (PC 2) principal components were sufficient to explain the total variance of 99.99 %. It was observed that no solvent systems possessed similar type of inhibitory properties. Thus all the results suggested that the ethanolic extract is the best solvent for extraction in terms of neuroprotective capacities of H. fastigiata extract (Fig. 6a, b).

Fig. 6
figure 6

a PCA analysis representing different extraction solvents for antioxidant capacities of H. fastigiata. Group I represents ethanolic extract (2) and ethyl acetate extract (3); Group II represents aqueous extract (1); Group III represents petroleum ether (4) extract. b PCA analysis representing different extraction solvents for neuroprotective capacities of H. fastigiata. Group I represents ethanolic extract (2) and ethyl acetate extract (3); Group II represents petroleum ether extract (4); Group III represents ethyl acetate extract (3); Group IV represents aqueous extract (1)

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Conclusion

Screening of different plant resources have achieved great successes in the past for finding potential neuroprotective compounds, such as the AChE inhibitor galanthamine. The present study revealed the utility of in vitro plantlets as a potential source of bioactive xanthones providing neuroprotective and antioxidant capacities. Because of the limited availability of this rare plant resource, the in vitro shoot culture system was established. The MS medium containing BA (1 mg/L) and kinetin (0.1 mg/L) was found to be most efficacious for in vitro propagation, with a growth index of 0.9, producing 15 ± 2 shoots per explant with shoot lengths of 2.7 ± 0.3 cm after 30 days of sub-culture. The total biomass yield achieved at the time of sub-culture was 25 ± 3 mg fresh weight (after 60 days). Three major xanthones were detected in shoot cultures of H. fastigiata. When compared, compound 3 showed the highest AChE inhibitory property along with inhibition of MAO-B, while compounds 1 and 3 showed selective inhibition of MAO-A and MAO-B, respectively. Compound 2 showed the strongest antioxidant capacity along with inhibition of both MAO-A and MAO-B enzymes. It was interesting to note that all the compounds exhibited the reversible type of inhibitions. The inhibition of MAO-A by compound 2 in a reversible and selective manner would be beneficial in the treatment of resistant depression, which comprises 33–57 % of all cases of depression. All the compounds showed good antioxidant properties. The significant AChE and MAO inhibitory properties of H. fastigiata suggest the potential of this plant for alleviation of neurological disorders associated with the depletion of acetylcholine and monoamines (AD and PD, respectively). Thus, it can be concluded that shoot cultures of H. fastigiata are a good source of xanthone compounds which can be interesting candidates for the design of new antidepressant drugs. This may have possible implications for herbal-based dietary supplements in alleviating these neurological disorders.