Analysing Curcuma caesia fractions and essential oil for neuroprotective potential against anxiety, depression, and amnesia

Abstract

Scientific pieces of evidence support the pharmacological activity of Curcuma caesia for its antidepressant, analgesic, anticonvulsant and antioxidant effect. Here, we evaluate the bioactivity of essential oil and the various polarity-based solvent partitioned fractions obtained from Curcuma caesia for anti-amnesia, anxiolytic and antidepressant activities using Elevated plus maze and Morris water maze models. The cold maceration technique using methanol was adopted for extraction from dried powdered rhizomes and essential oil was extracted by hydrodistillation method. Partitioning of the methanolic extract based on solvent polarity by hexane, ethyl acetate, and methanol was continued, followed by column chromatography of the ethyl acetate fraction. Suspensions were prepared for fractions (dissolved in distilled water) and essential oil (dissolved in tween 20) at 200 mg/kg and 400 mg/kg after acute toxicity study and were orally administered to Wistar albino female rats after the orientation of hypoxia by sodium nitrite (50 mg/kg) and amnesia by scopolamine (1 mg/kg). Behavioural observations, biochemical and histopathological examinations were carried out for all the treated groups. Diazepam (12 mg/kg) and galantamine (3 mg/kg) were used as standard drugs for this study against hypoxia and amnesia. Data acquired from behavioural, biochemical (acetylcholinesterase, myeloperoxidase, superoxide dismutase, reduced glutathione, catalase) and histopathological studies have illustrated that fraction II acquires highly significant memory-enhancing, anxiolytic and antidepressant effects. Rest fractions (I and III) and essential oil showed moderate efficacy. In prospects, identification of active molecules from the most active fraction (fraction II) and further studies on a molecular basis would substantiate its specific mechanism of neuroprotective action.

Introduction

Amnesia leads to memory impairment, which causes in the perturbation of day-to-day activities. The major factors causing amnesia are genetic, chemical, environmental, functional, oxidative stress, family history of Alzheimer's disease, head injury, or neurological causes. Oxidative stress (OS) is irrevocably synchronized with numerous foremost pathogeneses of Alzheimer’s disease that include Aβ-induced neurotoxicity, tau pathology, mitochondria dysfunction, and metal dyshomeostasis. In people suffering from amnesia, excessive production of reactive oxygen and reactive nitrogen species (RNS) causes brain injury through the breakdown of proteins, lipids, and nucleic acids. Oxygen consumption is more in brain tissues and that is why these tissues are more sensitive to oxidative stress and low level of antioxidants causes memory impairment (Francis et al. 1999; Zhao and Zhao 2013; Garry et al. 2015).

Hypoxia is a life-menacing situation as the third foremost basis of death worldwide wherein there is an inadequate release of oxygen in the tissues (Fonseca-Santos et al. 2015; Gueroui and Kechrid 2016). Hypoxia-ischemia is an instantaneous effect of hypoperfusion, that causes Aβ accumulation. Brain-based interleukin-1 (IL-1) regulates the cognitive function and excess IL-1 in the brain is congruent with memory loss and impaired learning. About 87% death of patients with hypoxic injury is because of cerebral ischemia that causes cognition and memory dearth (Chiu et al. 2012; Adhami et al. 2006; Garabadu and Krishnamurthy 2014).

Curcuma caesia Roxb. belongs to the Zingiberaceae family and is a perennial aromatic herb that possesses a bluish-black coloured rhizome that has a bitter taste with a pungent smell, commonly known as ‘Black Turmeric’ and locally known as ‘Kolaa Haladhee’ (in Assamese language). This ethnomedicinal plant is indigenous to northeast India along with central India and other parts of Asian countries (Das and Zaman 2013; Sahu et al. 2017). In the state of Manipur, India, the rhizome paste is traditionally used for the treatment of bruises and rheumatic pains (Sarangthem and Haokip 2010). The Adi tribes of Arunachal Pradesh drink the decoction of fresh rhizome for the treatment of diarrhea. Besides, the Khamti tribes of the Lohit district apply the paste of fresh rhizome in the treatment of snakebite and scorpion sting (Kagyung et al. 2010; Tag et al. 2007). Apart from antimicrobial activity, the essential oil of this plant rhizome has been reported to have antioxidant and acetylcholinesterase inhibitory activity (Das and Zaman 2013; Borah et al. 2020; Reenu et al. 2015). Besides, there are also scientific pieces of evidence that crude methanolic extract from this plant rhizome has smooth muscle relaxant, anxiolytic and antidepressant activities, which has a significance to its contribution towards protection against neurodegenerative disorders, as such, it was considered worthy to investigate and repurpose on the essential oil and different fractions from the rhizome part of this plant against in vivo anxiety, depression and amnesic action (Devi and Mazumder 2018; Arulmozhi et al. 2006; Lawand and Gandhi 2013; Pandey and Tripathi 2014).

Materials and methods

Sample collection and preparation

The rhizomes of Curcuma caesia (23 kg) were collected from Pasighat district of Arunachal Pradesh, India (latitude: 27° 32ʹ N to 29° 20ʹ N and longitude: 93° 48ʹ E to 95° 36ʹ E, elevation: 502 ft above mean sea level). The plant was authenticated at Botanical Survey of India, Eastern Regional Centre, Shillong, of specimen no. DU/HS/SB/2017/01 and Reference No. BSI/ERC/Tech/2017/114, dated: 30.05.2017.

Oil extraction

Fresh rhizomes of Curcuma caesia were cut and prepared in two batches of 6 kg each. Every two batches were individually put into a round bottom flask (1000 mL) filled with water and fitted to a Clevenger apparatus. The essential oil was extracted by hydrodistillation technique for 8 h (Mukunthan et al. 2014; Abu et al. 2017).

The phytochemical constituents of Curcuma caesia essential oil were interpreted by gas chromatography–mass spectrometry (GC–MS) on an Agilent 5921A gas chromatograph linked to a mass selective detector of Agilent 5975. The gas chromatograph was equipped with HP-5MS of measurement 30 m × 250 µm × 0.25 µm. The oven temperature was set at 50 °C for 1 min and raised at 20 °C/min to 200 °C/min, with the injector temperature at 280 °C. The sample measurement was of 10 µL volume, which was diluted to 1% with methanol and the mode of injection was by the splitless method. The carrier gas was helium. The spectra were scanned in the assortment from 50 to 550 m/z at 3 scans/s. The relative percentages were obtained for each phytochemical constituent of the essential oil concerning peak area % in sequence.

Phytoconstituents extraction

The process of cold maceration technique has been adopted as the extraction method with the second batch of rhizomes. After collection and cleansing, these rhizomes were immediately shade-dried (for 15 days) and pulverized to fine powdered material in a mechanical grinder and stored in an airtight container at 4 °C. For the extraction process, 6 kg of dried rhizomes was subjected to cold maceration soaked in 650 mL of methanol for seven days under refrigeration with repeated shaking. During the process, the crude powdered drug was soaked in methanol without changing the solvent until the drug was completely exhausted. After seven days, the filtration of the methanol extract was carried out by pressing through a muslin cloth and the filtrate obtained was concentrated. Bioactivity-guided fractionation of this extract was carried out in a separating funnel to separate all non-polar chemical constituents as there may be the scope of extracting both non-polar and polar compounds by methanol. Before fractionation, the extract was dissolved in distilled water and mixed well. Based on polarity, three different organic solvents viz., hexane (500 mL), ethyl acetate (500 mL), and methanol (500 mL) were used for partitioning of compounds from the crude methanolic extract. The fractions were concentrated in a rotary evaporator (purchased from Buchi R-300). The ethyl acetate fraction showed the maximum yield of 74% as compared with hexane fraction of 32% yield and methanol fraction of 47% yield; hence, for further bioactivity analysis, ethyl acetate fraction of the methanol extract was mixed with silica gel of 265 mesh size and loaded into the column. The eluents were partitioned using the following solvent ratios: hexane (100) < hexane: ethyl acetate (90:10) < hexane: ethyl acetate (80:20) < hexane: ethyl acetate (70:30) < hexane: ethyl acetate (60:40) < hexane: ethyl acetate (30:70) < ethyl acetate (100) < ethyl acetate: methanol (90:10) < ethyl acetate: methanol (80:20). The elutes were separated from these different solvents, collected in conical flasks and concentrated using a rotary evaporator (Zeb et al. 2014; Hossain et al. 2014). Total nine fractions were obtained which were combined based on similar spots revealed through thin-layer chromatography. After combining these nine fractions, three fractions were obtained. These three fractions and essential oil were evaluated for in vivo behavioural studies on the rat model.

Test animals

Female Wistar Albino rats weighing about 200–250 g were used for the investigation. These rats were sustained in polypropylene cages under average conditions of 12 hours light/dark cycle, temperature 27°C ± 2 °C, the relative humidity of 52% ± 3% with free access to standard pellet diet (Hindustan Lever Ltd., India), and water ad libitum. The animals were procured from M/S Chakraborty Enterprise, Kolkata, and the required approval for performing the animal experiment was obtained from the Institutional Animal Ethical Committee Registration no. 1706/GO/Re/S/13/CPCSEA and Approval No. IASST/IAEC/2018/09 dated 04/04/2018.

Toxicity study

The toxicity study was performed according to Organization for Economic Co-operation and Development Guideline No. 423 (OECD 423). Non-pregnant and nulliparous 10-week-old female Wistar Albino rats weighing 120 g of 75 nos. in whole, were chosen for toxicity study by random sampling technique and fasted overnight with free admittance of water before administration of the essential oil intraperitoneally and the three fractions (I, II and III) from Curcuma caesia orally. The oil and fractions were administered to (n = 5) overnight fasted rats at an initial dose of 5 mg/kg, 50 mg/kg, 100 mg/kg, 500 mg/kg and 2000 mg/kg body weight. Based on the survivability of the animals after 72 h, the optimum LD50 doses have been selected as 200 mg/kg (low dose) and 400 mg/kg (high dose) for the current in vivo investigations (OECD 2001). A schematic representation for the study of Hypoxia and Amnesia in rats is specified in Fig. 1a and b.

Fig. 1.

A Schematic representation for the study of a hypoxia and b amnesia in rats

Elevated plus-maze (EPM) test

The potential anxiolytic and antidepressant activities of the hypoxic and treated rats with the rhizome oil and fractions of Curcuma caesia were evaluated by the method as illustrated by Foyet et al. in 2012 (Foyet et al. 2012). The EPM consisted of four arms with two oppositely closed dark arms and two opposite open arms of length 54 cm and width 10 cm. The elevation level of the maze was 60 cm on top of the ground. The closed arms were pasted with walls of height 35 cm. Hypoxia was initiated in the rats' brains by the administration of sodium nitrite solution 50 mg/kg of body weight orally. The solution was fed by an oral gavage needle at a dosage of 5 mL/kg body weight for 30 days. Immediately after 30 days, on the 31st day, the rats were administered with the rhizome oil intraperitoneally and fractions (I, II and III) of Curcuma caesia orally, all at the doses of 200 mg/kg and 400 mg/kg body weight for 15 days. Similarly, the standard drug diazepam was fed orally to the rats at 1 mg/kg body weight. The treatments with tests and standard drugs were given 30 min before the maze test. Each animal was placed at the center of the maze in front of one of the enclosed arms. Entry into an arm would be defined as the point when the animal places all its four paws into the arm. The behavioural pattern (the time spent in open and enclosed arms) of each animal was videographed on Sony Cybershot DSC-RX100 20.2 MP Digital Camera with 3.6 X Optical Zoom for 5 min, 15 min, and 30 min, respectively. After each anxiolytic test, the maze was cleaned with cotton soaked in 70% ethanol and allowed to dry before the observations of the next animal (Pellow et al. 1985; Claudia et al. 2017; Handley and Mithani 1984).

Morris water maze (MWM) test

Spatial learning and memory were evaluated using MWM per the method illustrated by Nunez in 2008 (Nunez 2008). Amnesia was induced in the rats' brains by the administration of scopolamine (hyoscine butylbromide) in normal saline (1 mg/kg of body weight) intraperitoneally for a month. All the doses were given 30 min before the maze test and the rats were intraperitoneally injected with the rhizomes oil and orally fed with the fractions of Curcuma caesia at doses 200 and 400 mg/kg bodyweight for 15 days. Correspondingly the standard drug donepezil at 3 mg/kg bodyweight was fed orally to the rats. The MWM was enclosed with a circular opaque water pool of 27 °C with 180 cm diameter and 60 cm height. This spherical puddle was divided into East, West, North, South, Northeast, Northwest, Southeast, and Southwest with all quadrants being equally gaped by the side of the perimeter of the puddle. A red-coloured escape podium (12 cm diameter) was kept, 1.5 cm below the surface of the water. Initially, the rats were skilled to position the concealed puddle in five acquirement tryouts for successively five days. The rats were trained to reach the puddle within 300 s and were allowed to tolerate the escape puddle for 15 s. The escape puddle was placed in a stagnant point. Throughout each trial, the rats were placed in each quadrant to nullify the quadrant consequences. In each day's schedule of five trials, those rats which fell short to accomplish the task in less than 60 s on the 5th day of the trial were debarred from this experiment. During the schedule of the experiment, if any rat failed to locate the pubble in 60 s, then it was quietly guided to the platform and was allowed to stay there for 15 s. Retention trial was followed at the end of the experiment where the escape pubble was removed and the rat was allowed to swim for a period of 60 s interval. The behavioural pattern (escape latency) of each animal was measured and videographed on Sony Cybershot DSC-RX100 20.2 MP Digital Camera with 3.6 X Optical Zoom for 300 min, respectively, throughout each retention trial (Barua et al. 2015).

Brain histopathology

On the end day of each experiment, the conscious rats were decapitated utilizing a guillotine for craniotomy and the brains were removed within 30 s from the skull. The parietal bones were detached from the sagittal suture using forceps and the brains were taken out using a spatula. The brains were washed in 0.9% cold saline solution and placed overnight in a fixative containing 10% formalin. The tissues were dehydrated and then embedded in paraffin. After fixation after 48 h, paraffin-embedded blocks of brain tissue were cut serially into coronal slices of 2 to 5 µm thickness from the frontal region adjoining the cerebellar cortex and hippocampus and stained with hematoxylin and eosin (H and E) stain. The normal and injured Purkinje cells in the cerebellar cortex, the Ammon Horn regions in the hippocampus were examined under 10 × magnification using Zeiss trinocular microscope (Claudia et al. 2017; Chaudhari et al. 2017; Lee et al. 2018; Ekanem et al. 2016).

Measurement of neurotransmitter and oxidative stress markers

Instantaneously, the rats were sacrificed at the end of the experiment by decapitation and the brains that were collected were rinsed in ice-cold normal saline homogenized with ice-cold phosphate buffer (pH 8). These homogenates (10% w/v) were centrifuged at 12,000 rpm for 15 min at 4 °C and the supernatant was used for the estimation of acetylcholinesterase activity (AchE), reduced glutathione (GSH), catalase activity (Cat), superoxide dismutase (SOD) and myeloperoxidase activities (MPO).

Measurement of acetylcholinesterase activity

Measurement of AChE activity was determined by the spectrophotometric method through the technique as described by Ellman et al. in 1961 (Ellman et al. 1961). Homogenate of hippocampus region was blended with acetylthiocholine iodide which acts as a substrate. This reaction mixture was finally added up to a volume of 1.0 mL phosphate buffer (0.1 M at pH 7.4), 5’dithionitrobenzoic acid (DTNB) (7 mM). The breakdown of the substrate was estimated in a UV–Visible spectrophotometer (MultiscanGo, Thermo Fisher) at 412 nm and the results were expressed as units/mg of protein (Nampoothiri et al. 2015).

Measurement of glutathione levels

The intensity of reduced glutathione was measured spectrophotometrically by determination of dithiobis (2-nitro)-benzoic acid (DTNB) reduced by SH-groups, as described by Owens and Belcher (1965). Brain tissue homogenates of hippocampus region (sacrificed by cervical dislocation) were washed in 0.9% saline, soaked in filter paper, weighed, and stored at – 80 °C. Tissue fragments (200 mg) were thawed and homogenized on ice in 1 mL of homogenizing buffer (250 mM sucrose, 20 mM Tris–HCl, 1 mM dithiothreitol, pH 7.4), using glass-Teflon homogenizers. The homogenates were centrifuged at 75,000×g at 4 °C for 2 h. Supernatants were stored at − 80 °C. To 0.1 mL of different tissue samples, 2.4 mL of 0.02 M EDTA solution was added and kept on an ice bath for 10 min (for tissue protein precipitation). Then, 2 mL of distilled water and 0.5 mL of 50% TCA were added. This mixture was kept on ice for 10–15 min and then centrifuged at 3000×g for 15 min. After this, 1 mL of supernatant was taken and 2.0 mL of Tris buffer was added to it. Then, 0.05 mL of DTNB solution (Ellman’s reagent) was added and vortexed thoroughly. OD was read (within 2 to 3 min after the addition of DTNB) at 412 nm in UV–Vis Spectrophotometer of model no. SPECORD ® 50 PLUS Analytikjena against a reagent blank. Appropriate standards were run simultaneously. Data obtained from the standard curve were expressed as units/mg of protein.

Superoxide dismutase activity assays

Superoxide dismutase (SOD) activity was measured as per the method of Kakkar et al. (1984). Homogenate of hippocampus tissues was mixed with 4.0 mL of n-butanol and the blend was allowed to stand for 15 min followed by centrifugation for 15 min at 2500×g to split the butanol layer. The colour strength of the chromogen that develops a purple colour was measured at 560 nm in UV–Vis spectrophotometer model no. SPECORD ® 50 PLUS Analytikjena against butanol as a blank solution. The activity of superoxide dismutase was expressed in units/mg of protein.

$$I=\frac{C-T}{C}\times 100$$ 1

Catalase activity assays

Catalase activity of brain tissue homogenate in the hippocampus region was analyzed based on the ability of the enzyme to break down H₂O₂ (used as a substrate) by the method of Aebi (1984). About 100 μL of the tissue homogenate was taken in a tube containing 3.0 mL of (30 mM) H₂O₂ and blended with 0.1 mM of 1.0 mL phosphate buffer (pH 7.4). The time required for 0.05 optical density change was observed at 240 nm against a blank containing the enzyme source in H₂O₂ free phosphate buffer. The absorbance was noted at 240 nm in UV–Vis spectrophotometer model no. SPECORD ® 50 PLUS Analytikjena and after the addition of enzyme, Δt was noted till OD was 0.45. If Δt was longer than 60 s, the procedure was repeated with a more concentrated enzyme sample. Reading was taken at every 3 s interval. The activity of the enzyme was calculated using the molar extinction coefficient of 43.6 M/cm. A unit catalase activity is the amount of enzyme (E) that liberates half the peroxide oxygen from H₂O₂ solution of any concentration in 100 s at 25 °C. Catalase activity is expressed as follows:

$$\text{Units/mg}\text{of}\text{protein}=\frac{2.3}{\mathrm{\Delta }t}\times \frac{E_{\text{initial}}}{E_{\text{final}}}\times 1.63\times {10}^{-3}$$ 2

where E = optical density at 240 nm, 2.3 = factor to convert into the log, Δt = time required for a decrease in the absorbance.

Myeloperoxidase activity assays

Myeloperoxidase (MPO) enzyme activity was investigated by the technique illustrated by Bechard et al. 2009 (Florent-Bechard et al. 2009). The brain tissue homogenate was centrifuged at 12,000 rpm at 4 °C for 60 min. After the supernatant was collected, the reaction was initiated by mixing 660 mg/mL of O-phenylenediamine in 0.2 mM of 1.0 mL phosphate buffer at pH 7.4 and 300 mM H₂O₂ (Pulli et al. 2013)_. The absorbance was calculated at 492 nm in the UV–Vis spectrophotometer of model no. SPECORD ® 50 PLUS Analytikjena at an interval of 30 s for 2 min and calculated as follows:

$$B\times \frac{\text{sample}\text{dilution}\text{factor}}{\text{reaction}\text{time}}\times V$$ 3

where B = amount in nanomole of DTNB consumed, reaction time = (in minutes, at the time when stop mix was added), V = sample volume (mL) added to well.

Data analysis

Data were articulated as mean ± SEM. The analysis was carried out using Microsoft Excel 2007 and Kruskal–Wallis One-way analysis of variance (ANOVA) followed by post hoc least significant difference (LSD) test with statistical significance of p < 0.05.

Results

Yield and physical characteristic of essential oil

The essential oil yielded in the amount of 14 mL from 12 kg of fresh rhizome after hydrodistillation. The oil was volatile with pale yellow colour and was stored in an amber coloured glass vial at 4 °C before further analysis. The yield of fraction I was 5.11 g, fraction II was 7.4 g and fraction III was 8 g from 63 g of crude extract (extracted from 11 kg of dried rhizomes).

Result of the toxicity study

The toxic consequences of fraction I, fraction II, fraction III, and essential oil were observed on Wistar Albino rats (200 g) and it was established that all the five animals survived up to a dose of 2000 mg/kg. In 72 h, no skin irritation, such as paw itching, mutilation in food and water consumption, behavioural changes, such as convulsions, paw licking, locomotory abnormalities, or death, were noticed. Based on the toxicity studies, the doses have been predetermined as 200 mg/kg (low dose) and 400 mg/kg (high dose) for further investigations on amnesic and hypoxic rat models.

Effect of essential oil and fractions on elevated plus maze

In 5 min experimental trial, the fraction II at a high dose of 400 mg/kg produced significantly better antidepressant and anxiolytic effects demonstrated on elevated plus-maze as reflected with the increase in the number of entries and time spent on the open arm and decrease in the number of entries and time spent on the enfolded arm as compared with control and standard groups. The sodium nitrite-induced hypoxic group demonstrated 15.00 ± 2.88 s on the open arm and 288.00 ± 1.42 s on the enfolded arm. The time spent on the open arm for fraction II (400 mg/kg) was 168.00 ± 2.00 s whereas, on the enfolded arm, was 68.67 ± 1.33 s in contrast with control of 197.33 ± 1.76 s on the open arm and 36.00 ± 1.67 s on the enfolded arm and standard drug diazepam of 167.67 ± 2.33 s as time spent on open arms and 31.00 ± 2.08 s on enfolded arms significantly (p < 0.0001). Rats administered with fraction II in low dose (200 mg/kg) illustrated an interval of 115.00 ± 2.88 s on open arms and 117.00 ± 1.52 s on enfolded arms significantly (p < 0.0001). Similarly, an essential oil also illustrated its potency as an antidepressant and anxiolytic with its effect on rats as time spent on the open arm in low dose (200 mg/kg) as 55.00 ± 2.88 s and on enfolded arms was 169.00 ± 2.08 s. The essential oil in high dose (400 mg/kg) illustrated the period on the open arm as 71.67 ± 1.67 s and on enfolded arms at 158.00 ± 2.00 s. Their significance level was p < 0.0001. Consequently, in fraction I and fraction III, the time spent on the open arm was relatively less. In fraction I (200 mg/kg), the time spent on the open arm was 50.67 ± 2.96 s and on enfolded arms was 218.00 ± 4.40 s. In the case of fraction I (400 mg/kg), the time spent on the open arm was 65.00 ± 2.88 s and on enfolded arms was 175.00 ± 2.88 s. Fraction III (200 mg/kg) demonstrated the time spent on the open arm was at 85.00 ± 2.88 s and on enfolded arms at 159.33 ± 1.76 s, Fraction III (400 mg/kg), the time spent on the open arm was 106.67 ± 2.88 s and on enfolded arms was 110.67 ± 2.33 s significantly (p < 0.0001). In Fig. 2a, the time spent on open/enfolded arms in all treated and control groups has been demonstrated.

Fig. 2.

Graphical representation (mean ± S.E.M.) of anxiolytic and antidepressant behaviour on elevated plus maze; a bar plot representing the time spent on open/enfolded arms in hypoxic, all treated and control groups. The significance for all treated groups was p < 0.0001 as compared with both negative and positive controls; b bar plot representing the number of entries on open/enfolded arms in hypoxic, all treated and control groups. The significance for all treated groups was p < 0.05 as compared with both negative and positive controls. All values have been expressed as mean ± S.E.M., with a significant level of all treated groups, using One-way ANOVA followed by ‘t’—test in Graphpad Prism 7

The number of entries in open and enfolded arm for fraction II at a high dose of 400 mg/kg was 4.46 ± 0.29; 0.66 ± 0.33 concerning control of 5.33 ± 0.33; 0.66 ± 0.33 and diazepam was 5.67 ± 0.33 with no entries on the enfolded arm. At a low dose of 200 mg/kg, fraction II showed an entry for 3.67 ± 0.33 on the open arm and 0.67 ± 0.33 on the enfolded arm. However, the negative group showed 1.33 ± 0.33 on the open arm, and on the enfolded arm was 2.67 ± 0.33. In essential oil (200 mg/kg), number of entries on the open arm was 2.67 ± 0.67; on the enfolded arm was 1.33 ± 0.33, whereas in high dose (400 mg/kg), number of entries on the open arm was 2.73 ± 0.26 and on the enclosed arm was 1.33 ± 0.33. Both fraction II and essential oil showed a significance of p < 0.05. Fraction I (200 mg/kg) showed 1.33 ± 0.33 number of entries on the open arm; on the enclosed arm was 2.67 ± 0.33 and fraction I (400 mg/kg) showed 2.8 ± 0.2 no. of entries on the open arm; on the enclosed arm was 1.33 ± 0.33. In fraction III (200 mg/kg), number of entries on the open arm was 1.67 ± 0.33; on the enclosed arm was 1.33 ± 0.33 and in fraction III (400 mg/kg), number of entries on the open arm was 2.6 ± 0.23 and on the enclosed arm was 1.67 ± 0.67. The number of entries on open/enfolded arms in all treated and control groups has been established in Fig. 2b.

Effect of essential oil and fractions on AChE level of hypoxic rats

The AChE intensity was measured in the cerebral cortex region of the rat brain for all treated and control groups. It was estimated that the AChE level in the sodium nitrite hypoxic group was 26.48 ± 0.57 units/mg of protein which was significantly more (p < 0.05) than the control group of 13.89 ± 0.23 units/mg of protein concentration. In fraction II-treated groups of low (200 mg/kg) and high (400 mg/kg) doses, the AChE level was 19.12 ± 0.87 units/mg of protein and 17.74 ± 2.36 units/mg of protein. In case of essential oil of low (200 mg/kg) and high (400 mg/kg) doses, the AChE level was 21.11 ± 1.3 units/mg of protein and 17.80 ± 0.39 units/mg of protein. Fraction I (200 mg/kg) and (400 mg/kg) demonstrated 20.86 ± 0.30 units/mg of protein; 19.91 ± 0.90 units/mg of protein and fractions III (200 mg/kg) and (400 mg/kg) demonstrated 21.05 ± 0.57 units/mg of protein; 15.44 ± 0.33 units/mg of protein with respect to diazepam at 15.25 ± 0.13 units/mg of protein. The significant level of all treated groups is p < 0.05 and has been represented as a Heat map in Fig. 3, respectively.

Fig. 3.

Heat map representation of biochemical markers, where X-axis indicates all the treated groups of hypoxic rats and Y-axis indicates the biochemical markers from 1 to 5, where 1: AChE, 2: MPO, 3: SOD, 4: Red.GSH, 5: Catalase. The significance for all treated groups was p < 0.05. All values have been expressed as mean ± S.E.M., with a significant level of all treated groups, using One-way ANOVA followed by ‘t’—test in Graphpad Prism 7

Effect of essential oil and fractions on MPO level of hypoxic rats

The MPO concentration was measured in the cerebral cortex region of the rat brain for all treated and control groups. In the sodium nitrite hypoxic group, the MPO level was 254.35 ± 1.99 units/mg of protein which was significantly more (p < 0.05) than the control group of 77.96 ± 1.51 units/mg of protein concentration. In the hypoxic group treated with fraction II at low (200 mg/kg) and high (400 mg/kg) doses, the MPO intensity was 209.61 ± 4.19 units/mg of protein and 181.07 ± 4.07 units/mg of protein. In the case of the hypoxic group treated with essential oil at low (200 mg/kg) and high (400 mg/kg) doses, the MPO level was 216 ± 2.6 units/mg of protein and 190.94 ± 1.74 units/mg of protein. Fraction I (200 mg/kg) and (400 mg/kg) established its effect at a concentration 201.68 ± 2.41 units/mg of protein; 141.38 ± 1.00 units/mg of protein and fractions III (200 mg/kg) and (400 mg/kg) illustrated that 193.05 ± 1.65 units/mg of protein; 156.79 ± 2.8 units/mg of protein with respect to diazepam at 88.42 ± 1.98 units/mg of protein. The significance level of all treated groups was p < 0.05 and has been represented as a Heat map in Fig. 3, respectively.

Effect of essential oil and fractions on SOD level of hypoxic rats

The SOD concentration was estimated in the cerebral cortex region of rat brain for all groups and found that in sodium nitrite hypoxic group the SOD level was 41.67 ± 0.68 units/mg of protein which was significantly more (p < 0.05) than control group of 32.30 ± 0.27 units/mg of protein level. In hypoxia + fraction II group at low (200 mg/kg) and high (400 mg/kg) doses, the SOD intensity was 33.23 ± 0.09 units/mg of protein and 29.88 ± 0.13 units/mg of protein. In case of hypoxia + essential oil at low (200 mg/kg) and high (400 mg/kg) doses, the SOD concentration was 38.35 ± 0.68 units/mg of protein and 34.73 ± 0.30 units/mg of protein. In hypoxia + fraction I (200 mg/kg) and (400 mg/kg), its effective concentration was 38.94 ± 0.39 units/mg of protein; 36.85 ± 0.32 units/mg of protein and in fractions III (200 mg/kg) and (400 mg/kg), the SOD concentration was 35.91 ± 0.42 units/mg of protein; 34.02 ± 0.17 units/mg of protein with respect to diazepam at 28.44 ± 0.11 units/mg of protein. The significance for all treated groups was p < 0.05 and has been represented as Heat map in Fig. 3, respectively.

Effect of essential oil and fractions on Red. Glut. (GSH) level of hypoxic rats

The GSH amount was anticipated in the cerebral cortex region of the rat brain for all groups and initiated that in sodium nitrite-induced hypoxic group the GSH level was 5.38 ± 0.04 units/mg of protein which was significantly more (p < 0.05) than the control group of 3.45 ± 0.03 units/mg of protein level. In Nano₂ + fraction II group at low (200 mg/kg) and high (400 mg/kg) doses, the GSH intensity was 3.74 ± 0.06 units/mg of protein and 3.32 ± 0.16 units/mg of protein. In case of Nano₂ + essential oil at low (200 mg/kg) and high (400 mg/kg) doses, the GSH concentration was 3.67 ± 0.07 units/mg of protein and 3.57 ± 0.04 units/mg of protein. In Nano₂ + fraction I (200 mg/kg) and (400 mg/kg), its effective concentration was 4.60 ± 0.04 units/mg of protein; 4.11 ± 0.06 units/mg of protein and in fractions III (200 mg/kg) and (400 mg/kg), the GSH concentration was 4.22 ± 0.13 units/mg of protein; 3.79 ± 0.05 units/mg of protein with respect to diazepam at 3.49 ± 0.02 units/mg of protein. The significance for all treated groups was p < 0.05 and has been represented as a Heat map in Fig. 3, respectively.

Effect of essential oil and fractions on Catalase level of hypoxic rats

The catalase level was measured in the cerebral cortex region of the rat brain for all groups and initiated that in sodium nitrite-induced hypoxic group, the catalase level was 545.78 ± 3.58 units/mg of protein which was significantly at a reduced level (p < 0.05) than the control group of 765.89 ± 1.15 units/mg of protein level. In Nano₂ + fraction II group at low (200 mg/kg) and high (400 mg/kg) doses, the catalase activity was 715.89 ± 1.13 units/mg of protein and 687.65 ± 2.4 units/mg of protein. In case of Nano₂ + essential oil at low (200 mg/kg) and high (400 mg/kg) doses, the catalase activity concentration was 630.54 ± 1.12 units/mg of protein and 677.47 ± 1.04 units/mg of protein. In Nano₂ + fraction I (200 mg/kg) and (400 mg/kg), its effective concentration was 647.97 ± 1.61 units/mg of protein; 667.95 ± 3.72 units/mg of protein and in fractions III (200 mg/kg) and (400 mg/kg), the catalase concentration was 652.91 ± 1.14 units/mg of protein; 672.77 ± 1.84 units/mg of protein with respect to diazepam at 760.43 ± 1.16 units/mg of protein. The significance for all treated groups was p < 0.05 and has been represented as a Heat map in Fig. 3, respectively.

Brain histopathology

Histopathological assessment of normoxia, hypoxia, and treated groups has been carried out in hematoxylin–eosin stain and examined under 40 × magnification in Zeiss trinocular microscope. The Photomicrographic section of the hippocampus region in the normoxic group shows the regular configuration of cortical neurons and pyramidal cells as shown in Fig. 4a; the hypoxic group exemplifies the presence of inflammatory cells surrounding the blood vessels accompanied by cellular edema, neuronal degeneration, and autolysis following cell death as depicted in Figures Fig. 4b. In fraction II-treated group of low dose (200 mg/kg), a slight recovery of pyramidal cells' injury could be seen along with minor inflammatory changes; however, in high dose (400 mg/kg), the injured pyramidal cells of Ammon’s horn were restored to normal levels and no inflammatory cells could be noticed similar to diazepam as shown in Fig. 4c1, c2 and d. The essential oil in low-dose treatment (200 mg/kg) showed slight positive changes to the degenerated neuronal cells, however, in high-dose treatment (400 mg/kg), illustrated moderate restoration of damaged cells to normal levels as is depicted in Fig. 4e1 and e2. The other fractions, i.e fraction I and fraction III, in low doses (200 mg/kg) showed no positive changes to the injured pyramidal cells and cellular edema could also be seen with no signs of improvement as seen in Fig. 4f1 and g1. In high doses (400 mg/kg), both fractions I and III showed temperate transformations of nuclear morphology as shown in Fig. 4f2 and g2), respectively.

Fig. 4.

Histopathological Representation of rat brain; a normoxic group showing regular configuration of cortical neurons and pyramidal cells; b the hypoxic group exemplifying the presence of inflammatory cells surrounding the blood vessels accompanied by cellular oedema, neuronal degeneration and autolysis following cell death; c1 fraction II-treated group of low dose (200 mg/kg) a slight recovery of pyramidal cells’ injury could be seen along with minor inflammatory changes; c2 in Fraction II-treated group of high dose (400 mg/kg), the injured pyramidal cells of Ammon’s horn were restored to normal levels and no inflammatory cells; d the histopathological representation of standard treated groups (diazepam) have shown configurations similar to normoxic group as all neuronal abnormalities have been restored to normal levels; e1 essential oil in low dose treatment (200 mg/kg) showed slight positive changes to the degenerated neuronal cells; e2 essential oil in high-dose treatment (400 mg/kg) illustrated moderate restoration of damaged cells to normal levels; f1 fraction I in low dose (200 mg/kg) showed no positive changes to the injured pyramidal cells and cellular oedema could also be seen with no signs of improvement; f2 in high dose (400 mg/kg), fraction I showed temperate transformations of nuclear morphology; g1 fraction III in low dose (200 mg/kg) showed no positive changes to the injured pyramidal cells and cellular oedema could also be seen with no signs of improvement; g2 In high dose (400 mg/kg), fraction III showed temperate transformations of nuclear morphology

Effect of essential oil and fractions on escape latency in Morris water maze results

There was a decrease of escape latency significantly (p < 0.05) on the 15th day experimental trial in rats of drug-treated groups than in a scopolamine-induced amnesic group of 54.47 ± 0.35 s. In the control group, the escape latency period was 6.29 ± 0.65 s. Fraction II in high dose (400 mg/kg) illustrated an equal frequency of escape latency period as control with 6.72 ± 0.38 s, in low dose (200 mg/kg) with 8.41 ± 0.30 s and galantamine-treated group (standard) with 5.31 ± 0.20 s, respectively. Besides, fraction III in both low (200 mg/kg) and high (400 mg/kg) doses indicated a better escape latency effect with 18.61 ± 0.61 s and 15.67 ± 0.46 s than fraction I at low dose (200 mg/kg) with 40.31 ± 0.69 s, at high dose (400 mg/kg) with 30.69 ± 0.34 s and essential oil (200 mg/kg) with 28.11 ± 0.72 s; (400 mg/kg) with 22.93 ± 2.68 s. These results have been illustrated in Fig. 5.

Fig. 5.

Graphical representation of (mean ± S.E.M.) escape latency of all fractions and oil in Morris Water Maze. The bar plot significance for all treated groups was p < 0.05 as compared with both negative and positive controls. All values have been expressed as mean ± S.E.M., with significant level of all treated groups, using One-way ANOVA followed by ‘t’—test in Graphpad Prism 7

Effect of essential oil and fractions on AChE level of amnesic rats

The AChE intensity was measured in the cerebral cortex region of the rat brain for all treated and control groups. It was estimated that the AChE level in the scopolamine-induced amnesic group was 31.5 ± 1.57 units/mg of protein which was significantly more (p < 0.05) than the control group of 15.19 ± 0.10 units/mg of protein level. In fraction II-treated groups of low (200 mg/kg) and high (400 mg/kg) doses, the AChE level was 22.29 ± 1.00 units/mg of protein and 19.04 ± 0.79 units/mg of protein. In case of essential oil of low (200 mg/kg) and high (400 mg/kg) doses, the AChE level was 25.21 ± 1.14 units/mg of protein and 21.15 ± 0.16 units/mg of protein. Fraction I (200 mg/kg) and (400 mg/kg) demonstrated 23.96 ± 2.92 units/mg of protein; 19.76 ± 1.94 units/mg of protein and fractions III (200 mg/kg) and (400 mg/kg) demonstrated 23.44 ± 1.90 units/mg of protein; 19.03 ± 0.21 units/mg of protein with respect to galantamine at 13.88 ± 0.20 units/mg of protein. The significant level of all treated groups is p < 0.05 and has been represented as a Heat map in Fig. 6, respectively.

Fig. 6.

Heat map representation of biochemical markers, where X-axis indicates all the treated groups of amnesic rats and Y-axis indicates the biochemical markers from 1 to 5, where 1: AChE, 2: MPO, 3: SOD, 4: Red.GSH, 5: Catalase. The significance for all treated groups was p < 0.05. All values have been expressed as mean ± S.E.M., with a significant level of all treated groups, using One way ANOVA followed by ‘t’—test in Graphpad Prism 7

Effect of essential oil and fractions on MPO level of amnesic rats

The MPO concentration was measured in the cerebral cortex region of the rat brain for all treated and control groups. In the scopolamine-induced amnesic group, the MPO level was 145.95 ± 3.00 units/mg of protein which was significantly more (p < 0.05) than the control group of 75 ± 2.35 units/mg of protein level. In the amnesic group treated with fraction II at low (200 mg/kg) and high (400 mg/kg) doses, the MPO intensity was 108.87 ± 4.69 units/mg of protein and 90.73 ± 2.97 units/mg of protein. In the case of the amnesic group treated with essential oil at low (200 mg/kg) and high (400 mg/kg) doses, the MPO level was 121.74 ± 1.42 units/mg of protein and 125.25 ± 5.57 units/mg of protein. Fraction I (200 mg/kg) and (400 mg/kg) established its effect at a concentration 119.06 ± 0.60 units/mg of protein; 116.76 ± 3.18 units/mg of protein and fractions III (200 mg/kg) and (400 mg/kg) illustrated that 118.95 ± 0.74 units/mg of protein; 113.62 ± 2.9 units/mg of protein with respect to galantamine at 67.64 ± 1.24 units/mg of protein. The significance level of all treated groups was p < 0.05 and has been represented as a Heat map in Fig. 6, respectively.

Effect of essential oil and fractions on SOD level of amnesic rats

The SOD concentration was estimated in the cerebral cortex region of rat brain for all groups and found that in scopolamine-induced amnesic group where, the SOD level was 47.09 ± 0.40 units/mg of protein which was significantly more (p < 0.05) than control group of 34.67 ± 0.49 units/mg of protein level. In amnesia + fraction II group at low (200 mg/kg) and high (400 mg/kg) doses, the SOD intensity was 37.46 ± 0.68 units/mg of protein and 31.83 ± 0.62 units/mg of protein. In case of amnesia + essential oil at low (200 mg/kg) and high (400 mg/kg) doses, the SOD concentration was 41.06 ± 0.91 units/mg of protein and 37.55 ± 0.69 units/mg of protein. In amnesia + fraction I (200 mg/kg) and (400 mg/kg) its effective concentration was 42.69 ± 0.82 units/mg of protein; 38.30 ± 0.55 units/mg of protein and in fractions III (200 mg/kg) and (400 mg/kg) the SOD concentration was 41.92 ± 0.67 units/mg of protein; 37.5 ± 1.21 units/mg of protein with respect to galantamine at 30.88 ± 0.73 units/mg of protein. The significance for all treated groups was p < 0.05 and has been represented as Heat map in Fig. 6, respectively.

Effect of essential oil and fractions on Red. Glut. (GSH) level of amnesic rats

The GSH amount was anticipated in the cerebral cortex region of the rat brain for all groups and initiated that in scopolamine-induced amnesic group where the GSH level was 7.77 ± 0.05 units/mg of protein which was significantly more (p < 0.05) than the control group of 3.21 ± 0.09 units/mg of protein level. In scopolamine + fraction II-treated group at low (200 mg/kg) and high (400 mg/kg) doses, the GSH intensity was 5.04 ± 0.21 units/mg of protein and 3.52 ± 0.08 units/mg of protein. In case of scopolamine + essential oil at low (200 mg/kg) and high (400 mg/kg) doses, the GSH concentration was 6.18 ± 0.04 units/mg of protein and 5.86 ± 0.04 units/mg of protein. In scopolamine + fraction I (200 mg/kg) and (400 mg/kg), its effective concentration was 6.43 ± 0.04 units/mg of protein; 5.9 ± 0.06 units/mg of protein and in fractions III (200 mg/kg) and (400 mg/kg), the GSH concentration was 5.64 ± 0.23 units/mg of protein; 4.77 ± 0.15 units/mg of protein with respect to galantamine at 2.95 ± 0.03 units/mg of protein. The significance for all treated groups was p < 0.05 and has been represented as a Heat map in Fig. 6, respectively.

Effect of essential oil and fractions on Catalase level of amnesic rats

The catalase level was measured in the cerebral cortex region of the rat brain for all groups and initiated that in scopolamine-induced amnesic group, the catalase level was 529.01 ± 1.19 units/mg of protein which was significantly at a reduced level (p < 0.05) than the control group of 768.6 ± 2.0 units/mg of protein level. In scopolamine + fraction II group at low (200 mg/kg) and high (400 mg/kg) doses, the catalase activity was 690.85 ± 1.78 units/mg of protein and 738.95 ± 3.17 units/mg of protein. In case of scopolamine + essential oil at low (200 mg/kg) and high (400 mg/kg) doses, the catalase activity concentration was 580.68 ± 4.26 units/mg of protein and 608.82 ± 0.64 units/mg of protein. In scopolamine + fraction I (200 mg/kg) and (400 mg/kg), its effective concentration was 584.35 ± 1.3 units/mg of protein; 604.48 ± 1.6 units/mg of protein and in fractions III (200 mg/kg) and (400 mg/kg), the catalase concentration was 571.81 ± 1.69 units/mg of protein; 632.39 ± 2.02 units/mg of protein with respect to galantamine at 764.13 ± 2.86 units/mg of protein. The significance for all treated groups was p < 0.05 and has been represented as a heat map in Fig. 6, respectively.

Brain histopathology

Cerebral cortex histopathology was observed for all diverse groups tarnished in hematoxylin–eosin stain and examined under 40 × magnification in Zeiss trinocular microscope. The Photomicrographic section of this region in the normal group showed the normal texture of neurons as shown in Fig. 7a; the amnesic group exemplified the presence of neurofibrillary tangles and abnormal cell morphology accompanied by perivascular edema as depicted in Fig. 7b. The essential oil in low-dose treatment (200 mg/kg) showed slight positive pathological changes to the cortex cells, however, high-dose treatment (400 mg/kg) demonstrated moderate restoration of degenerated cells to regular texture as shown in Fig. 7c1, c2. In fraction II-treated group of low dose (200 mg/kg), partial gliosis could be observed with minor inflammatory changes, conversely in a high dose (400 mg/kg), diffused gliosis with no inflammatory cells could be noticed, and degenerated cells were reinstated to moderate level. Similarly in galantamine, all features could be restored to normal as depicted in Fig. 7d1, d2 and e. Fraction I and fraction III in low doses (200 mg/kg) showed minor alterations to the injured cells and irregular cellular outline still found as seen in Fig. 7f1 and g1. In high doses (400 mg/kg), both fractions I and III showed moderate transformations of cerebronuclear morphology as shown in Fig. 7f2 and g2, respectively.

Fig. 7.

Histopathological Representation of rat brain; a normal group showing normal texture of neurons; b the amnesic group exemplifying the presence of neurofibrilliary tangles and abnormal cell morphology accompanied by perivascular oedema; c1 essential oil in low dose treatment (200 mg/kg) showed slight positive pathological changes to the cortex cells; c2 essential oil in high-dose treatment (400 mg/kg) demonstrated moderate restoration of degenerated cells to regular texture; d1 in fraction II-treated group of low dose (200 mg/kg) a partial gliosis could be observed with minor inflammatory changes; d2 in fraction II-treated group of high dose (400 mg/kg), diffused gliosis with no inflammatory cells could be noticed and degenerated cells were reinstated to moderate level; e in standard drug (galantamine)-treated group, all features could be restored to normal; f1 fraction I in low dose (200 mg/kg) showed minor alterations to the injured cells and irregular cellular outline; f2 in high dose (400 mg/kg), fraction I showed moderate transformations of cerebronuclear morphology; g1 fraction III in low dose (200 mg/kg) showed minor alterations to the injured cells and irregular cellular outline; g2 in high dose (400 mg/kg), fraction III showed moderate transformations of cerebronuclear morphology

Discussion

The central cholinergic coordination occupies a vital position in cognition and its deficit causes a decline in learning and memory for humans and animals. Conservation of acetylcholine levels facilitates in the restoration of memory. Cerebral hypoxia causes brain ischemia, stroke and is correlated with memory impairment and dementia. Oxidative stress causes neurodegeneration. Chemicals like scopolamine and sodium nitrite amplify oxidative stress that is involved in the pathogenesis of neurodegenerative diseases (Puri et al. 2014). In our study, we have investigated the alterations of memory impairment and neuronal homeostatic imbalance experimented by induction of amnesia by scopolamine and anxiolytic, antidepressant effects conducted through stimulation of sodium nitrite-induced hypoxic brain ischemia. Scopolamine is chemically (+)-Hyoscine of molecular formula C₁₇H₂₁NO₄. This drug is a non-selective muscarinic antagonist, which after administration obstructs the cholinergic signaling and causes short- and long-term memories as well as cognitive dysfunctions. At the research level, it has been observed that this chemical induces an amnesic effect in animal models. It favourably hinders muscarinic receptors for acetylcholine levels and decreases the levels of acetylcholine. It further impairs learning and memory causing depression of the cerebral cortex (Barua et al. 2015; Kulkarni et al. 2010; Kheirbakhsh et al. 2018). Sodium nitrite is chemically also known as nitrous acid of molecular formula NaNO₂ or sodium salt. Induction of this element causes intoxication to the brain nerve cells, thereby increasing the susceptibility of our central nervous system to hypoxic conditions. The mechanism involves the binding of this inorganic salt to hemoglobin, thus forming methemoglobin. This byproduct diminishes the capacity of hemoglobin to carry oxygen. The reactive nitrogen species generated by this mechanism causes damage to the neuronal cells and by this means one may suffer from anxiety and depression (Foyet et al. 2012; Claudia et al. 2017). These effects are correlated with the decline in the levels of catalase, elevation in AChE, MPO, SOD, and Red. GSH levels. Synthetic drugs, such as cholinesterase enzyme inhibitors (galantamine and donepezil), anti-epileptic agents (felbamate), calcium channel blockers (nifedipine), anxiolytics and antidepressants (alprazolam, chlordiazepoxide, clonazepam, diazepam, flurazepam; fluoxetine and nootropic agents (piracetam), slow the succession of this disease, but lack to cure completely. However, with our perception of cellular and biochemical occurrences observed in the cognitive brain, we have expanded our research to herbal technologies to develop toxic-free molecules. Since ancient ages, ethnomedicinal plants have been used as a natural remedy for diverse ailments. Their high therapeutic values appeal to our society to discover and develop a fundamental cure for all different ailments including neurodegenerative diseases. In our study, we have repurposed the therapeutic value of Curcuma caesia rhizome fractions and essential oil as a neuroprotective. Arulmozhi et al. (2006) has reported that the methanolic extract of this plant exhibited a significant smooth muscle relaxant activity on Guinea pig and Rabbit at a dose of (50–800 µg/mL) and has produced relaxation in trachea tissues pre-contracted with carbachol (Arulmozhi et al. 2006). Besides, this plant has shown a smooth muscle relaxation effect in presence of various receptor antagonists, such as propranolol, glibenclamide, 2′, 5′-dideoxyadenosine, α-chymotrypsin, L-NNA, and methylene blue. Karmakar et al. (2011a, b) have studied the antioxidant, antidepressant, analgesic, and anticonvulsant effect of the methanolic extract of rhizome of Curcuma caesia. The researcher had carried out the antidepressant activity using a forced swim and tail suspension test. Further, he had carried out the analgesic and anti-convulsant effect using an actophotometer and rotarod apparatus. These efficacies were evaluated with the crude methanolic extract nevertheless; the phytoconstituents responsible for such activities and their mechanism of action are still to be investigated (Karmakar et al. 2011a, b). Based on these shreds of evidence, we have determined to repurpose the whole rhizome part including the essential oil of Curcuma caesia based on bioactivity-guided fractionation for its efficacy as an antiamnesic and antihypoxic drug. For this reason, we have chosen two main models of study: elevated plus maze for anxiety, depressant related studies, and Morris water maze for learning and memory-related behaviour studies. Rodents were selected as subjects for determining the anxiolytic and antidepressant effects of the therapeutic drugs. Independent of training, in the EPM, the anxiolytic and antidepressant effects of the animals were evaluated based on the time spent and number of entries in open and enfolded arms. When the animals were placed at the central position, they started exploring the field towards either an open maze passage or enfolded maze passage. Those with brain abrasions explored the enfolded arm passage for a significantly longer duration. The animals were treated with essential oil and three different fractions (fraction I, fraction II and fraction III) at 200 mg/kg and 400 mg/kg bodyweights. The standard drug chosen for the study was diazepam. As with diazepam, fraction II at high dose illustrated an amplification in the exploration and number of entries in the open arms in a dose-dependent manner. Similarly, essential oil at high dose also demonstrated an increase in the number of entries and time of exploration in open arms. In both these fractions, the time spent and number of entries in enfolded arms were appreciably decreased signifying a reduction in their levels of stress than the negative control group. Fractions I and III at high doses also demonstrated an increase in number and time of open arm entry; however, the anxiolytic effectiveness is less than diazepam. Diazepam belongs to the benzodiazepines class of anxiolytic drugs which stimulate an increase in number of entries and time spent in open arms. Fraction II and essential oil illustrate the behavioural result similar to diazepam and therefore we can articulate that the fractions of Curcuma caesia may enhance the GABA receptors response in the central nervous system (Karmakar et al. 2011a, b).

Spatial learning and memory for the fractions were evaluated using the MWM task on rodents. This method was developed by Richard G. Morris in 1984 and therefore this task has been named after him and considered as a gold standard for neurological studies. In this, animals were placed in a pool of water that was made opaque with powdered non-fat milk where the rodents were allowed to swim and reach the hidden escape point. The water pool was made opaque with the goal of not allowing the animals to sense the escape point through its wisdom of perceptions. Before this task, the animals were trained for 5 days on a clear pool of water to make them recognizable with the task and following 5 days of attainment trial (Nunez 2008; Allen 2018).

The animals were treated with essential oil and three different fractions (fraction I, fraction II and fraction III) at 200 mg/kg and 400 mg/kg bodyweights. The standard drug chosen for the study was galantamine. Analogous with galantamine at the low dose, fraction II at high dose illustrated an intensification in the escape latency period and cumulative path length in a dose-dependent manner than the scopolamine-induced amnesic group that depicted an extended escape latency period and cumulative path length. Similarly, essential oil at high dose also demonstrated an increase in escape latency period and cumulative path length. Fractions I and III at high doses also demonstrated a moderate increase in escape latency period and cumulative path length, nevertheless, their memory-enhancing effectiveness was not as much of galantamine. Galantamine belongs to the tertiary alkaloidal group of drugs isolated from the flowers and bulbs of Galanthus caucasicus and Galanthus woronowii, which belong to the Amaryllidaceae family. This drug belongs to the cholinesterase inhibitors that diminish the central cholinergic neurotransmission (Tewari et al. 2018; Scott and Goa 2000). Fraction II and essential oil exemplified the behavioural result similar to galantamine, and therefore we can articulate that the fractions of Curcuma caesia acts as an allosteric modulator of nicotinic acetylcholine receptors in the central nervous system. Further, the fractions were assessed for in vivo antioxidant markers and the neurotransmitter acetylcholine.

Acetylcholine neurotransmitter which signals through metabotropic muscarinic and ionotropic nicotinic receptors induces synaptic plasticity, transmission and maintains the co-ordination of neurons. This transmission has lapsed through acetylcholinesterase enzyme which hydrolyzes and breaks down acetylcholine neurotransmitter into acetate and choline before it arrives at the receptors. Cholinergic dysfunction is correlated with neurodegenerative diseases and hence its inhibition would help in restoring the cholinergic functions (Picciotto et al. 2012). In our study, essential oil, fraction II, and fraction III have shown a high inhibition of acetylcholinesterase enzyme in both amnesia and hypoxia rodent models and thus progresses cognitive function. Since the induction of sodium nitrite and scopolamine enhances oxidative injury to the brain, there is a significant amplification of MPO, SOD, GSH levels and decline of Catalase oxidative markers. These markers play an important role in balancing oxidative stress. Upon pretreatment with fractions II and III at 400 mg/kg illustrated a significant refurbishment of these markers and reduced more free radicals analogous to the standard drugs. Other fractions viz., essential oil, fraction 1 also showed a moderate restoration of these markers to a normal level as in the control group. The high to moderate restoration properties of these markers by all the fractions are correlated with its free-radical-scavenging potential. Besides, brain histological studies show that all the fractions of this plant have regenerated the hypoxic and injured neuronal cells of the cerebral cortex and hippocampus indicating its neuroprotective action.

The plausible grounds for the anxiolytic and antiamnesic consequences for these fractions are the presence of phytoelements in these fractions. The isolated pure compounds as such may have a limitation of being less potent than the partitioned fraction. The partitioning of these fractions based on solvent polarities gives us a suggestion that the fractions have polyphenols, terpenoids, alkaloids, and tannins that are responsible for antiamnesic and antihypoxic consequences. The plant rhizome has beneficial as a high antioxidant, carminative, anti-diarrheal, diuretic, antiemetic, pain reliever, and in skin disorders (Kagyung et al. 2010). Due to its acetylcholinesterase inhibitory activity and free-radical-scavenging properties, this plant may have a prospective role in the facilitation of pre- and post-synaptic cholinergic transmission. As compared to the accessible synthetic drugs, the use of these toxic-free phyto-elements of this plant as therapeutic antioxidants has proved to be effective in treating such neurological disorders that show the way towards oxidative stress. Hence, Curcuma caesia is a valued and rare medicinal herb that needs to be preserved for its neuroprotective efficacy.

Conclusion

The authors would like to suggest from the report that rhizome fractions and essential oil of Curcuma caesia may have significant positive therapeutic effects on anxiety, depression and could enhance the memory as assessed by behavioural investigation through Elevated plus maze and Morris water maze. Further increase of acetylcholine levels could be attained in the cholinergic cells of the brain through inhibition of acetylcholinesterase enzyme and regeneration of neuronal cells as represented in brain histological studies. Identification of the active molecule responsible for these activities and further studies on a molecular basis would substantiate its specific mechanism of neuroprotective action.

Abbreviations

Nos.

Numbers

RNS

Reactive Nitrogen Species

OECD

Organization for Economic Co-operation and Development

EPM

Elevated plus maze (EPM)

MWM

Morris water maze

cm

Centimeter

GSH

Reduced glutathione

Cat

Catalase activity

SOD

Superoxide dismutase

MPO

Myeloperoxidase

DTNB

5′ dithionitrobenzoic acid

mM

MilliMolar

EDTA

Ethylenediamine tetra acetic acid

TCA

Tricholoacetic acid

g

Gram

O.D.

Optical density

UV–Vis spectrophotometer

Ultraviolet–visible spectrophotometer

H₂O₂

Hydrogen peroxide

SEM

Standard error of the mean

Funding

This research work received fund from Council of Scientific and Industrial Research (CSIR), HRDG, New Delhi under CSIR Direct SRF Scheme Vide File No. 09/857 (0010)/2018-EMR-I.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.