Phytochemical and Spectral analysis of Argoclavine; A potent Psychoactive substance present in the seeds of Argyreia nervosa plant

 

A. V. Deokar*, P. V. Bhosle

Shreeyash Institute of Pharmacetical Education and Research, Aurangabad, Maharashtra, India.

*Corresponding Author E-mail: pallavi.1230@gmail.com, arundhati.deokar@gmail.com

 

ABSTRACT:

There are several medicinal plants having various medicinal properties is being widely used in Ayurveda which contain phytoconstituents. These phytoconstituents are beneficial to manage allied symptoms in psychosis andto treat abnormal condition of the mindthereby promoting mental well being. Argyreia nervosa (L.f) the ancient traditional medicinal plant belonging to the convolvulaceae family having prominent, psychedelic and neuro-pharmacological actions on our body. Argyreia nervosa seeds contain hallucinogens including ergot alkaloids such as ergine and Argoclavine and a naturally occurring lysergic acid amide. The current study describes pharmacognostic and phytochemical screening on Seeds of Argyreia nervosa. Argoclavine was extracted from the seeds and then characterised by different spectroscopic techniques like UV, IR, LC-MS. The results of the present study will create a way for the invention of novel herbal medicines for several ailments by using Argyreia nervosa seeds.

 

KEYWORDS: Ergoline alkaloids, Convolvulaceae, Hallucinogens, Argoclavine, Neuropsychatric diseases.

 

 


INTRODUCTION:

In Ayurvedic medicine, every part of number of medicinal plants used including the seed, leaf, bark and root have usage as they possess a broad-range of pharmacological activities such as antimicrobial, antidiarrhoeal, hepatoprotective, anticonvulsant, antioxidant, aphrodisiac, immuno modulatory, analgesic and anti-inflammatory activity1. One such plant is Argyreia nervosa (L.f) Sweet, which have various medicinal properties is widely used in Ayurveda, the ancient traditional medicinal system in India belonging to Convolvulaceae family. The seeds contains the main neuro psychoactive substances such as ergoline alkaloids that are used as a hallucinogen and have been used traditionally in a number of diseases in India.2,3

 

Ergoline alkaloids are main constituents of plants belonging to Convolvulaceae family and clavicipitaceous fungi (Ascomycota). Biosynthetic origin of ergoline alkaloids in family Convolvulaceae is unknown, to study these about 12 endophytic fungi and one epibiotic fungus were isolated from an ergoline alkaloid-containing Convolvulaceae family plants4,5

 

One study have revealed that Invitro and invivo cultivation on intact plants gave no evidence that the endophytic fungi are responsible for the accumulation of ergoline alkaloids in Convolvulaceae family plants5. But epibiotic clavicipitaceous fungus is equipped with the genetic material to synthesize these compounds. This fungus attacks on back side of leaf in favorable environmental conditions and synthesize ergoline alkaloids which later on translocated from leafs to seed and stored in seeds6. Several observations strongly indicate that this plant-associated fungus and its unidentified relatives occurring on different Convolvulaceae plants are responsible for the isolated occurrence of ergoline alkaloids in Convolvulaceae.7

 

Ergine a class of ergoline alkaloid is present in Argyreia nervosa seed8 A study reported stereoisomers of ergine to be found in the seeds at a concentration of 0.325% of dry weight9.  A more recent study reported presence of ergometrine, lysergol, lysergic acid and other alkaloids that contribute to its prominent, psychedelic and neuro-pharmacological actions on our body10.

 

So in present study, the pharmacognostic and phytochemical characteristics of seeds of the plant Argyreia nervosa was done and attempt was done to isolate the most potent phytoconstituent; Agroclavine. Which is then characterized by different spectroscopic techniques like UV, IR, LC-MS. The results of the present study will create a way for the invention of novel herbal medicines for several ailments by using Argyreia nervosa seeds.

 

METHODS:

a)    Pharmacognostic evaluation:

1.1. Plant material collection and authentication:

Seeds of Argyreia nervosa were collected in the month of September from the Botanical garden of Government College of pharmacy, Amravati. The plant was identified at the Department of Botany, VMV College, and Amravati and the study was carried out in the year 2019.

 

1.2.  Morphology:

The fresh matured seeds were selected to study the morphological characteristics. The various parameters like; type of cotyledons, appearance, shape, size – length and height, surface characteristics, were studied. Results shown in table- 1.3, fig. -1.1 and 1.2

 

1.3. Microscopy:

In TS the elements observed under microscope were captured with the help of camera and for powder microscopy shade dried and milled powder of seeds of Argyreia nervosa was used.

 

The elements observed under microscope were captured with the help of camera and displayed in the results, as shown in the figure-1.3 and 1.4

 

2. Extraction of seeds:

Only fully matured fruits of Argyreia nervosa were collected in the month of august-september and dried in shade after fully drying seeds are removed from fruit and powdered and stored in a well closed container for further use.

 

Argyreia nervosa seeds contain about 0.3-1% ergot-alkaloids. Ergine (d-lysergic acid amide), isoergine (l-lysergic acid amide), ergometrine, lysergol, iso-lysergol, elymoclavine and chanoclavine, Agroclavine are present11,12

2.1. Selection of solvent:

Ergoline alkaloid present in seeds are basically classified into three types as water-soluble amides of lysergic acid, the water-insoluble ergopeptines (i.e. ergo peptides), and the clavine group13. These alkaloids are insoluble in water and soluble in organic solvents, such as diethyl ether, chloroform and 1,2-dichloroethane.

 

2.2. Continuous hot percolation:

The alkaloid extraction was carried out by continuous hot percolation method using acetone as a solvent. For extraction 23grams of coarsely powdered seeds placed in thimble Soxhlet apparatus. Extraction was carried out for 3 and 1/2 hours for a total of 11 siphons next, extract concentrated and mixed with 100ml of water. With citric acid the pH is adjusted to 3. The solution is defeated 8 times with 50ml DCM, shaken vigorously without an emulsion forming. A light tan water layer and an amber DCM layer was produced after first defat. By the 4th defeat both layers are colourless. Total 8 defats are carried out for removal of toxins, pH is adjusted to 9 with sodium carbonate. With 50ml DCM the water layer is extracted 5 times. Freebase LSA (and other alkaloids) in it again distilled with DCM until it becomes 50ml and then evaporate the rest of the DCM in a shallow pan. The extract becomes light tannish yellow. Once dry it amounts to 72mg (about a 0.3 % yield). At this point the extract is probably 90% pure alkaloids14.

 

2.3. Alkaloid Extraction (short method) by Maceration:

Type-1:

Weigh 100g finely ground seeds and add NaHCO3, from these mixture alkaloids are extracted by maceration in ethyl acetate for one day. After one day filter the whole content. Obtained ethyl acetate extract is extracted with tartaric acid solution. After basification of the extract with NaHCO3, it was again extracted with ethyl acetate. Ethyl acetate layer upon evaporation to dryness in vaccum yield alkaloids. Repeat this procedure until there is no more residues.

 

Type -II:

Weigh 100g finely ground seeds, add 100ml petroleum ether and let soak about two days. Filter, discard petroleum ether and let seeds dry. In next step procedure is repeated by using 100ml methanol. After filtration, extract is evaporated in vacuum. The residual yellow oil contains the alkaloids.

 

The percentage yield was calculated for individual extraction methods and results are shown in table-1.6

 

2.4. Phytochemical Evaluation of seed extract:

Phytochemical evaluation of seed extract was carried out for analysis of various phytoconstituents.15


Table 1.1-Phytochemical evaluation of seed extract

Sr. No.

Tests

Observations

1

Carbohydrate

 

Violet ring at the junction of two liquids

Molish’s test (General test)

Fehling’s test (Reducing sugar)

First yellow than red ppt.

2

Proteins

 

Violet or pink colour

Biuret test (General test)

Millon’s test

White ppt. Turn brick red ppt on heating

3

Fat and oils

Permanently stained with oil

Filter paper test

4

Glycosides

 

Pink to red colour.

Leagal’s test

Borntrager’s tes

Pinkish or red colour to ammonia layer

Cyanogenic glycoside

sodium picrate test

Filter paper turns brick red or maroon

5

Vitamins

 

Blue colour is produced immediately.

Vitamin A

Vitamin c

Yellow colour turns blue.

6

Saponins

 

Persistent foam

Foam test

7

Alkaloids

 

Orange brown ppt.

Dragendroff’s test

Mayer’s test

Cream coloured ppt.

8

Tannins and Phenolic compounds

 

White ppt

Lead acetate solution

 


Results of analysis was shown in table-1.7

 

3. Identification of compounds:

3.1. Identification of seeds extract by TLC:

Scheme of TLC was selected for identification of compounds16

 

Solvent system –chloroform: methanol (8:2)

Detection (Visualization) – By spraying with van urks reagent.

 

Agroclavine purchased from Sigma Aldrich was used as marker compound for identification of Agroclavine. Also from literature survey it was found that agroclavine shows blue coloured bands at Rf value 0.38

 

Results of analysis shown in figure-1.5

 

3.2. Isolation of Phytoconstituents:

For isolation of argoclavine, Preparative thin layer chromatography technique is used17,18,19. Results of percentage yield shown in table no -1.8

 

4. Characterization of the isolated compound:

4.1. UV spectroscopy:

The UV spectroscopy is useful to measure the number of conjugated double bonds and also aromatic conjugation within the various molecules. From literature survey it is found that agroclavine shows absorbance at 225nm, 283 nm and 293nm, when UV spectra of isolated component was taken it also shows absorbance at same wavelength showing that isolated component was agroclavine.

 

Preparation of sample –The isolated compound was dissolved in methanol and absorbance was determined by UV spectrophotometer.

Blank-methanol

Scanned at Wavelength range-200 to 400nm

The presence of conjugation was determined by UV spectroscopy.

The UV spectrum is shown in the figure -1.6

 

4.2. IR spectroscopy:

IR spectroscopy is used for detection of functional group in the isolated compounds. The IR spectrum of isolated compound shows prominent peaks at 3643 which indicates the presence of indole nucleus, peak at 3236 shows NH stretching, presence of (C=C) was shown by presence of peak at 1605 and 1445. Peak at 2922 shows Alkyl C-H Stretch, Alkane C-H bonds are fairly ubiquitous and therefore usually less useful in determining structure. The IR spectrum is shown in the figure 1.7

 

4.3. Liquid chromatography -Mass spectrometry analysis:

LC-MS or HPLC-MS is a hyphenation of liquid chromatography with mass spectrometry. Within a minute the mass spectrum of the substance obtained by direct extraction into HPLC-MS20,21. To increase stability and for better extraction in polar solvents during extraction seeds were treated with sodium bicarbonate which results in formation of molecular ion peak at 452 m/e value with base peak at 329m/e value. The molecular ion shows many fragments at 400-452m/e value, among which m/e value 452 mass unit is significant. According to literature survey fragmentation of argoclavine shows molecular ion peak at m/e value 241.

 

Results of analysis was shown in Figure 1.8, 1.9 and 2.0

 

RESULTS:

Pharmacognostic evaluation:

Macroscopy:

 

Table 1.2- Morphology of Seeds

Features

Inference

Colour

Brownish white

Shape

More or less triangular

Length/height

0.5 to 0.75 cm long up to 5 mm broad

Apex/base

Two concave sides and one convex side

Surface

Outer surface is glabrous

Texture

Hard and not easily breakable

Odour and taste

The odor is not characteristic while taste slightly astringent

 

 

Figure 1.1- Fruits of Argyreia nervosa Figure 1.2- Seeds of Argyreia nervosa

 

Microscopy:

 

Figur 1.3-T. S of seed

 

Fig 1.4 - Powder Microscopy

 

Extraction and Phytochemical Investigation

 

Table-1.3: Evaluation of extraction of methods seeds

Sr. No

Method of extraction

% yield

1

Continuous hot percolation method with the help of Soxhelet apparatus

 8.10%

2

Maceration method

 Type 1- By using ethyl acetate

 3.41%

Type 2- By using petroleum ether and methanol

 5.83%

 

Phytochemical evaluation of Argyreia nervosa seeds extract

 

Table 1.4: Phytochemical evaluation of Argyreia nervosa seeds extract:

Sr. No

Test

Hot percolation

Maceration

Type-I

Type-II

1

Carbohydrate

-ve

-ve

+ve

Molish’s test

Barfoed’s test

-ve

-ve

+ve

2

Proteins

 -ve

 +ve

+ve

Biuret test

Millon’s test

 -ve

+ve

+ve

3

Fat and oils

 

-ve

 

-ve

 

+ve

Filter paper test

 4

Glycosides

 

-ve

 

-ve

 

+ve

Leagal’s test

Borntrager’s test

-ve

-ve

-ve

 5

Saponin

 

-ve

 

+ve

 

+ve

Foam test

 6

Alkaloids

 

-ve

 

+ve

 

+ve

Dragendroff’s test

Mayer’s test

-ve

+ve

+ve

Wagner’s test

-ve

+ve

+ve

 7

Tannins

 

-ve

 

-ve

 

ve

Lead acetate solution

 

Identification of seed extract:

 

Figure 1.5 -TLC of seed extracts showing presence of Agroclavine

Solvent system- Chlorform : Methanol (8:2)

Detection- By spraying with Van urks reagent blue colored .

Band of agrovlavine at rf value 0.38 was observed.

 

1.    Isolation of Agroclavine:

Table 1.5: Percentage yield of Agroclavine in Argyreia nervosa seeds extract

Sr.No

Method of extraction

% yield of Agroclavine

1

Continuous hot percolation method with the help of Soxhelet apparatus

 0.011%

2

Maceration method

 Type 1- By using ethyl acetate

 0.04 %

Type 2- By using petroleum ether and methanol

 0.06%

 

Characterisation of Isolated Component:

1. UV spectra 2. IR spectra of Agroclavine:

 

Figure 1.6 -U. V spectra of isolated compound.

 

 

Figure 1.7- I.R spectra of isolated compound

 

3. LC-MS of isolated component:

HPLC spectrum of isolated compound shows peak at retension time of 9 minutes mass spectrum of same was recorded by MS-MS spectrometry.

 

 

Figure 1.8- HPLC-MS spectra of isolated compound (IIT, Bombay)

 

Figure 2.0-MS spectra of isolated compound showing further fragmentation pattern of isolated compound (Courtesy-IIT, Bombay)

 

Further fragmentation of same produce fragment with m/e 203, m/e 184, m/e 197.

 

DISCUSSION:

Non-Hawaiians recently discovered hallucinogenic properties of Argyreia nervosa in the 1960s Chemical composition of its seeds is nearly identical to some of the species in Convolvulaceae family.

 

Among other species of convolvulaceae family, seeds of Argyreia contain the highest concentration of psychoactive compounds in the entire family. To produce psychedelic effects seeds of the plant can be consumed Argyreia nervosa seeds contain 0.3-1% ergot-alkaloids by weight. Seeds are rich in Ergine (d-lysergic acid amide), isoergine (l-lysergic acid amide), ergometrine, lysergol, isolysergol, elymoclavine and chanoclavine. Reduction products of d-lysergic acid 6, 7 Lysergol and elymoclavine are also present in seeds.

 

Recent findings indicate that psychedelic drugs can alter the neurotransmitter release and provides neuroprotection. So they may have therapeutic potential for the treatment of neurodegenerative diseases like parkinsons disease. Also other neuropsychiatric disorders such as anxiety, chronic depression like depression associated with end stage cancer etc to get faster recovery.

 

Taking this study data in consideration; So, The current study describes pharmacognosticand phytochemical screening on seeds of Argyreia nervosa and further study was conducted to isolate agroclavine a class of ergoline alkaloid called argoclavinefrom the seeds of Argyreia nervosa plant. Which is then characterised by different spectroscopic techniques like UV, IR, LC-MS.

 

The pharmacognostic and phytochemical evaluation of the seeds is essential because there are different plant species available in convolvulceae. family which contains ergoline alkaloids; Hence It is possible to mix genuine Argyreia nervosa seeds with those from other plants, so to avoid adulteration and to get easy in selection pharmacognostic and phytochemical screening was carried out.

 

The LC-MS spectroscopic analysis resulted to be an effective tool for the identification of phytoconstituents in any plant parts. So this technique was used for the determination of molecular weight of agroclavine present in the Argyreia nervosa seeds. The selectivity of this technique allows the characterization of compound not only by their pseudo molecular ions but also by their specific fragmentation patterns. All results were noted and the conclusion was made.

 

ACKNOWLEDGEMENTS:

Author would like to thank to the Principal, Government college of Pharmacy, Amravati for providing necessary facilities for this research and SAIF IIT, Bombay, for spectral analysis.

 

PLANT AUTHENTICATION:

The plant was authenticated by Dr, Prabha Bhogaonkar, Botanist Government Vidarbha Institute of Science and Humanities.

 

ETHICS APPROVAL AND CONSENT TO PARTICIPATE:

The author declares that the work did not involve humans or animals which required ethical approval or consent to participate.

 

CONFLICTS OF INTEREST:

No Conflicts of Interest Regarding the Publication of this Manuscript.

 

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Received on 08.04.2021        Modified on 13.05.2021

Accepted on 03.06.2021  ©AandV Publications All right reserved

Res. J. Pharmacognosy and Phytochem. 2021; 13(3):109-114.

DOI: 10.52711/0975-4385.2021.00018