Characterization of coumarins from Ipomoea mauritiana Jacq by LC-APCI-MS/MS analysis and evaluation of its anti-amnesic activity

Abstract

Background

Ipomoea mauritiana is one of the source plants of Vidari, an Ayurvedic drug used as Medhyarasayana (rejuvenating drug). In this current study, coumarins were separated from tuberous root of I. mauritiana and characterization of the coumarin fraction was done by LC-MS/MS analysis by atmospheric pressure chemical ionization method. Anti-amnesic activity was evaluated against scopolamine induced amnesia in Wistar rats.

Results

Mass spectroscopic characterization of coumarin fraction directed to the tentative identification of coumarins such as 7-hydroxy-6-methoxy coumarin, 7-hydroxycoumarin, 5-methoxy-6,7-furanocoumarin, 5,7-dimethoxycoumarin, and 6-hydroxy-7-methoxy-4-phenylcoumarin. Aqueous extract of I. mauritiana at a dose of 100 and 200 mg/kg showed significant anti-amnesic activity against scopolamine-induced changes in step through latency and working memory errors.

Conclusion

The findings of the study showed that I. mauritiana is a rich source of coumarins and possessed significant anti-amnesic activity. The study concluded the scientific basis of using I. mauritiana as rejuvenating drug in Ayurveda.

Graphical abstract

Background

Ipomoea mauritiana Jacq. (IM) is a medicinal plant (Convolvulaceae) used in Ayurveda and Folk medicine. It is one of the source plants of Vidari, an Ayurvedic drug which is a component of about 50 Ayurvedic formulations including Chyawanprash [1]. The roots are sweet, cooling in action, appetizer, galactagogue, rejuvenating, stimulant, carminative, and tonic. It is also used in emaciation, enteric fever and spermatorrhea [2,3,4]. It contains phytochemicals such as taraxerol, taraxerol acetate, β-sitosterol, scopoletin, 7-O-β-d-glycopyranosyl scopoletin and caffeoyl glucose [4, 5].

Alzheimer's disease is a progressive neurological disorder that mostly affects the elderly population. Learning and memory impairment as the most distinguishing expression of dementia could be induced chemically by scopolamine, a cholinergic antagonist. Cholinergic deficits are neuropathological occurrences that are consistently associated with memory loss and are correlated with the severity of Alzheimer’s disease [6,7,8].

Chemically coumarins are benzopyrones and are fairly ubiquitous in the plant kingdom; more than 3400 naturally occurring coumarins have been identified [9, 10]. Recently, coumarin chemistry has attracted much attention due to diverse biological and pharmacological properties such as anticoagulant, antibacterial, antifungal, antiprotozoal, insecticidal, fungicide, antimycobacterial, antimutagenic, HIV protease inhibition, monoamine oxidase (MAO) inhibition, and anti-inflammatory activity. Number of evidences also shows the implication of coumarin in the inhibition of acetylcholinesterase enzyme because the planarity and aromaticity of the coumarin ring is the key structural feature that allows its interaction easily with PAS through possible π-π stacking [11, 12].

In the present study, coumarins were separated from the tuberous roots of I. mauritiana, and their structural characterization was done by LC/MS analysis. Aqueous extract was screened for its antiamnesic activity against scopolamine induced amnesia in Wistar rats.

Methods

Chemicals and instruments

High performance thin layer chromatography (HPTLC) profiling was done on pre-coated silica gel 60F₂₅₄ TLC plate (Merck, India). The mobile phase was standardized as toluene:ethyl acetate:methanol in the ratio 8:2:1. The chromatogram was developed in a saturated chromatographic chamber (Camag, Switzerland), and the developed plate was visualized under 366 nm. LC/MS analysis was conducted on Agilent 6520 accurate mass Q-TOF LC/MS coupled with Agilent LC 1200 equipped with Extend-C18 column of 1.8 μm, 2.1 × 50 mm. The APCI source was operated with following settings in positive mode: drying gas (nitrogen) flow 8 L/min; nebulizer pressure 40 psig; drying gas temperature 325 °C; capillary voltage + 3000 V; fragmentor volt 125 V; Oct Rf Vpp 750 V. Gradient elution was performed with water/0.05% formic acid (solvent A) and acetonitrile (solvent B) at a constant flow rate of 0.9 ml/min. Column temperature was maintained at 30 °C.

Plant material

Mature tubers of I. mauritiana were collected from Herb garden, Kottakkal, Kerala, India, and authenticated by Dr. K M Prabhukumar, Plant Systematics and Genetic Resources Division, Centre for Medicinal Plants Research (CMPR), Kerala, India, and a voucher specimen was deposited in CMPR herbarium.

Extraction of coumarins

Coumarins were separated by specific method. Briefly, 50 g of the material was extracted with methanol by soxhlet extraction method for 48 h. After evaporating the extract under reduced pressure, the residue was refluxed with 5% HCl for 2 h. After filtration two fractions were obtained, residue and aqueous—acid phase. The residue was extracted with methanol and evaporated (Fr 1) and aqueous—acid phase was extracted with diethyl ether and was evaporated (Fr 2). The fractions (Fr 1 and Fr 2) were kept under refrigerator until further analysis.

Acute oral toxicity study

Plant extracts

The pharmacological evaluation was done in aqueous extract. Two hundred fifty grams of the tubers of IM was extracted with water using soxhlet extraction method for 72 h. The final extract was concentrated to dryness under reduced pressure using rotary evaporator (Heidolph, Germany).

Target animals

The experiment was conducted on Wistar rats (females) weighing 147 to 204 g and aged 8 to 9 weeks obtained from the Animal House, J.S.S. College of Pharmacy, Ootacamund-Tamil Nadu. The rats were distributed into 5 groups with 6 animals in each group. The experimental procedures relating to the animals were authorized by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) (Approval No: JSSCP/IAEC/OT/Ph.D/Ph.Cology/06/2017-18) before starting the study and were conducted under the internationally accepted principles for laboratory animal use and care.

The extracts were prepared from a plant material having a high safety margin, and hence, it was decided to use 2000 mg/kg (limit test) for this study. The test item was prepared immediately prior to administration on respective treatment days. A quantity of 2 g of the test item was dissolved in distilled water, and the volume made up to 10 ml to get a test item concentration of 200 mg/ml. Homogeneity of the test item in the vehicle was maintained during treatment by constant stirring and mixing. The test substance was administered soon after preparation. The prepared test item solutions were administered once orally as gavage to the fasted (16–18 h) rats at the dose volume of 10 ml/kg b.wt. to deliver a dose of 2000 mg/kg b.wt. Food was offered about 3–4 h after dosing. Water was not withheld.

The treated rats were observed five times during day 1 (day of administration), i.e., at 30 min and four times at hourly (post-administration) intervals and once daily and, thereafter, for a total of 14 days. The clinical signs were recorded on all working days. The body weights of rats were recorded on test day 1 (pre-administration), day 8 (7 days post-administration), and day 15 (14 days post-administration). The rats were euthanized by using diethyl ether anesthesia and necropsied.

Evaluation of anti-amnesic activity

The test item was prepared immediately prior to administration on respective treatment days. The extract was prepared as solution in distilled water at concentration equal to 1/10th of the dose and administered at a dose volume of 10 ml/kg, b.wt. Animals were randomly divided into seven groups containing 6 each.

Group treatment

1Normal (vehicle 10 ml/kg, p.o.)

2Control (vehicle 10 ml/kg, p.o.)

3Rivastigmine (1.5 mg/kg., p.o.)

4IM (100 mg/kg., p.o.)

5IM (200 mg/kg., p.o.)

The above treatment was given for a period of 14 days, and a day before the memory test, all the animals were given appropriate training. On 14th day, 1 h after the respective treatments, scopolamine (3 mg/kg., i.p.) was administered to all the groups, except group 1, normal. Step down and step through latency were evaluated 45 min after the scopolamine administration to assess the memory, and the radial arm maze memory test was carried out on day

Step through latency was evaluated by the standard procedure. Each rat was individually placed in the bright part of a two-chambered apparatus for training. The door was closed once rat enters the dark chamber to prevent it from escaping. Later a foot shock (1 mA, 1 s) was applied through the grid floor, and the rat was then returned to the home cage.

Twenty-four hours later, testing was repeated by placing the animal again in the bright chamber. The latency period to enter the second darker chamber was measured. A prolonged latency indicates that the animal remembers that it has been punished and, therefore, does avoid the darker chamber.

Step down latency was assessed using a rectangular box (50 × 50 cm) with electrifiable grid floor and 35 cm fits over the block. The grid floor is connected to a shock device which delivers scrambled foot shocks. The experiment was conducted in three phases: (1) familiarization—the animal was placed on the platform, released after raising the cylinder, and the latency to descend is measured. After 10 s of exploration, it was returned to the home cage. (2.) Learning—immediately after the animal has descended from the platform, an unavoidable foot shock was applied (foot-shock 50 Hz; 1.5 mA; 1 s) and the animal is returned to the home cage, (3) retention test—24 h after the learning trial the animal was again placed on the platform, and the step-down latency was measured. The test was finished when the animal steps down or remains on the platform (cutoff time 60 s).

Radial arm maze test was performed in a eight arm radial maze. Animals were placed in wooden elevated eight-arm radial maze with the arms extending from a central platform 26 cm in diameter. Each arm is 56 cm long and 5 cm wide with 2 cm high rails along the length of the arm. The maze was well illuminated and numerous cues are present. Food pellets (reward) will be placed at the end of the arms. During the test, rats were well fasted to motivate them to run the maze. Animals were trained on a daily basis in the maze to collect the food pellets. The session was terminated after 8 choices, and the rat has to obtain the maximum number of rewards with a minimum number of errors.

The number of errors (entries to non-baited arms) will be counted during the session: reference memory error: Visit to unbaited arm more than once; working memory error: Visit to baited arm more than once.

Statistical analysis

For determination of significant intergroup differences of each parameter one-way analysis of variance (ANOVA) was carried out. Dunnet’s test was used for individual comparisons after significant ANOVA results. The differences with p < 0.05 were considered statistically significant. Graph pad Prism-6 software (Graph pad software, Inc., USA) was used for the statistical analysis.

Results

Chemical profiling

Chemical profiling of crude extract was done by HPTLC for the preliminary chemical characterization. HPTLC profile at 366 nm showed several bands with blue fluorescents. The presence of coumarins was confirmed by derivetizing with alcoholic KOH. Various intensified blue fluorescence bands were observed on UV-366 evaluation (Fig. 1).

Fig. 1

HPTLC analysis of IM extract

LC-MS analysis

Two separated coumarin fractions (Fr1 and Fr2) were subjected to LC/MS analysis in APCI positive mode. The total ion chromatogram (TIC) was extracted with molecular feature extraction (MFE) using Agilent Mass hunter software. The base peak chromatogram of fraction 1 and fraction 2 are presented as Figs. 2 and 3. On evaluating the various protonated ions, ms/ms analysis was conducted by selecting preferred precursor ion on the basis of abundance in m/z values. Mass fragmentation was achieved by collision-induced dissociation (CID) by assigning collision energy 4 eV/100 Dalton with an offset voltage of 6 eV. Consistency of fragments was confirmed by targeted ms/ms analysis with fixed collision voltage as obtained from auto ms/ms analysis. The tentative structure of coumarins was assigned based on mass fragmentation pattern in correlation with previously reported data [13, 14]. The coumarins identified by APCI-MS/MS analysis are presented in Table 1 and Fig. 4.

Fig. 2

LC-APCI-MS base peak chromatogram of fraction 1

Fig. 3

LC-APCI-MS base peak chromatogram of fraction 2

Table 1 APCI-LC-MS/MS analysis of coumarin fraction of IM

Fig. 4

Coumarins identified from IM

Acute oral toxicity studies

The acute oral toxicity results of IM extract on administration once orally to Wistar rats at a dose of 2000 mg/kg b.wt. are presented in Table 2.

Table 2 Body weight, body weight changes, and pre-terminal deaths

Based on the results of the acute oral toxicity (acute toxic class method) of I. mauritiana extract in Wistar rats, the LD₅₀ of the extract may be classified as GHS category 5 (LD 50 > 2000 mg/kg) as per OECD Guideline No. 423, December 2001.

Evaluation of anti-amnesic activity

Effect of test item on body weight

The effect of IM on body weight of the experimental animals is presented in Table 3. There was no significant difference in the body weights were observed with the treated groups when compared to control group (p > 0.05).

Table 3 Effect of IM extract on weekly body weight of Wistar rats

Effect of test items on step through latency

The effect of IM extracts on scopolamine induced amnesia in Wistar rats is presented in Table 4. The results showed that scopolamine administration (3 mg/kg., i.p.) has significantly decreased the step through latency in passive avoidance test in group 2, control animals when compared with group 1, normal animals (p < 0.05), indicating amnesic effect of scopolamine. Groups 4 and 5 animals treated with IM extract at a dose of 100 and 200 mg/kg, p.o., respectively, showed a significant dose dependent protection against scopolamine induced memory loss (p < 0.05).

Table 4 Effect of IM extract on scopolamine-induced amnesia in Wistar rats

Effect of test items on step down latency

The results of the study showed that scopolamine administration (3 mg/kg., i.p.) has significantly decreased the step down latency in passive avoidance test in group 2, control animals when compared with group 1, normal animals (p < 0.05), indicating amnesic effect of scopolamine. Groups 4 and 5 animals treated with IM extract at a dose of 100 and 200 mg/kg, p.o., respectively, show no significant protection against scopolamine induced memory loss (p > 0.05).

Effect of test item on reference and working memory errors in radial arm maze test

The results of the study showed that scopolamine administration (3 mg/kg., i.p.) has significantly increased reference and working memory errors in radial arm maze test in group 2, control animals when compared with group 1, normal animals (p < 0.05), indicating amnesic effect of scopolamine. Group 4 and 5 animals treated with IM extract at a dose of 100 and 200 mg/kg, p.o., respectively, show a significant protection against scopolamine induced working memory errors (p < 0.05) and a non-significant protection against scopolamine induced reference memory errors (p > 0.05).

Discussion

In Ayurveda, Medhya Rasayana is termed for rejuvenating drugs with numerous benefits specifically to improve memory and intellect. I. mauritiana is the one of the rejuvenating drugs mentioned in the Ayurvedic texts. The preliminary chemical screening of I. mauritiana was done HPTLC analysis. Crude alcoholic extract showed presence of coumarins, and the same was confirmed by derivetizing the chromatographic plate with alcoholic potassium hydroxide, a common TLC reagent for the easy identification of coumarins [15]. Coumarins are large group of natural compounds isolated from many plants and are still being reported each year. Many coumarins with remarkable biological activities such as anticoagulant, antiproliferative, anti-microbial, etc., have been reported from many medicinal plants [16,17,18,19].

Method has been optimized for the separation of coumarins from the crude extract. Two fractions were separated, and the same was subjected to tandem mass spectroscopic analysis. Mass fragmentation based on collision induced dissociation led to the identification of five coumarins from the two fraction of I. mauritiana. Learning and memory enhancement activity of coumarin like scopoletin has been reported earlier [20, 21].

The effect of aqueous extract of I. mauritiana was evaluated on scopolamine induced amnesia in Wistar rats. It has been established that the increased activity of acetylcholinesterase enzyme in brain decreases the acetylcholine levels leading to memory loss [22, 23]. Scopolamine, a muscarinic antagonist, has shown to improve the acetylcholinesterase activity, and it has also been reported that decreased muscarinic activity causes formation of β-amyloid plaques in brain leading to memory impairment in Alzheimer’s disease (AD) [24]. Moreover, increased acetylcholinesterase activity has also been reported to enhance the aggregation of β-amyloid proteins into more toxic amyloid plaques, worsening the condition especially in AD [25]. IM has shown to reverse scopolamine-induced amnesia imparted significant protection against Scopolamine induced memory loss

Conclusion

The present study was conducted to identify active principles of I. mauritiana and to evaluate its memory enhancing activity by phytochemical and pharmacological analyses. Preliminary chromatographic analysis of I. mauritiana extract showed presence of coumarins, and subsequent tandem mass spectroscopic characterization of the same led to the identification of five coumarins in which four are new report for this species. Anti-amnesic activity was evaluated against scopolamine-induced amnesia in Wistar rats. The result showed significant protection against scopolamine-induced working memory errors and a non-significant protection against scopolamine-induced reference memory errors. The outcome of the present study is a scientific validation of traditional knowledge.