Preliminary Phytochemical Screening of Leaves, Stems and Roots of wild Cannabis sativa

 

Komal Sharma, Rinu Rana*, M S Ashawat

Pharmacognosy Department, Laureate Institute of Pharmacy, Kathog, Himachal Pradesh-176031.

 

ABSTRACT:

Cannabis sativa Linn. (Cannabinaceae family) is traditional plant well known for its analgesic property. Cannabis is an indigenous plant of Himachal Pradesh. The aim of the present study is to check the phytochemicals present in leaf, stem and roots of wildly grown cannabis plant in the state of Himachal Pradesh. The study also includes morphological and microscopical studies of leaf, stem and roots of wildly grown cannabis plant. The preliminary phytochemical screening of cannabis shows the presence of alkaloids, tannins, phenolic compounds, proteins, amino acids, flavonoids and steroids.

 

 

 

INTRODUCTION:

Cannabis (Cannabis sativa Linn.) also known as Marijuana, is a well-known psychoactive annual dioecious flowering plant cultivated and used throughout the world as a source of many valuable phytoconstituents. It has been used traditionally as a source of fuel, paper, medicine and textiles.¹ Its origin is not clear but it is believed to be the first cultivated non-food crops. The temperate region of central Asia is thought to be its origin.² Due to morphological differences in various forms of Cannabis sativa (C. sativa), scientists has included other two species, Cannabis indica and Cannabis ruderalis as subspecies.³ In India, three herbal preparations of cannabis are popular known as Bhang, Ganja and Charas.⁴

 

The aromatic resin in the flowering tops of the cannabis contains chemical compounds having psychoactive activities.⁵ Scientists have discovered over 400 chemical compounds found in all parts of the cannabis plant.⁶   

 

Cannabinoids have a variety of effects on humans. The main constituent of C. sativa plant is tetrahydrocannabinol (THC). It has action on central nervous system and causes mild euphoria and hallucinations. Cannabidiol (CBD) has sedative and relaxant effect.⁷ The well-known side effects of utilizing cannabis with higher concentrations are nausea, dizziness, weakness, psychosis, dysphoria, anxiety, behavioral and mood changes, hallucinations and feelings of intoxication.⁸

 

Other cannabinoids present in the plant are cannabichromene (CBC), cannabiciclol (CBL), cannabigerol (CBG), cannabigerol monomethyl ether (CBGM), cannabielsoin (CBE), cannabinodiol (CBND), cannabitriol (CBT), dehydrocannabifuran, and cannabicitran.^9,10 It has been reported to be used in malignant brain tumors, neurological disorders (parkinson's and alzheimer's disease), multiple sclerosis, neuropathic pain, lennox-gastaut and dravet syndromes.¹¹

 

MATERIALS AND METHOD:

Collection of the plant samples:

Fresh plants of C. sativa were collected from the Beh area near by Badhal Thore, district Kangra from Himachal Pradesh. The plant is wild which is grown by its own.

 

Morphological studies:

The freshly collected leaves, stems and roots were thoroughly washed, and organoleptic characters were determined.

 

Microscopical studies:

The freshly collected leaves, stems and roots were thoroughly washed and cut into transverse sections (T. S.). The sections were treated with chloral hydrate solution and stained with phloroglucinol and hydrochloric acid (HCl) for the microscopic evaluation.

 

Phytochemical screening:

The leaves, stems and roots were washed; shade dried and powdered using a mechanical blender for the phytochemical screening.¹²

 

 

Chemical tests:

1: Test for carbohydrates:

Molisch’s test:

1-2 ml of Molisch’s reagent were added in 2-3ml alcoholic extract of cannabis leaves, stem and roots, and concentrated sulphuric acid (H₂SO₄) were added from side of the test tube, a violet ring was formed at the junction of the liquid.

 

2. Test for reducing sugar:

Fehling’s test:

1ml Fehling’s A and 1ml Fehling’s B solution were mixed and boiled for one minute. Equal volume of test solution was added. The mixture was heated in boiling water bath for 5-10 min. First a yellow then brick precipitate was formed.

 

3. Test for alkaloids:

Dragendroff’s test:

To few ml of alcoholic extract of C. sativa leaf, stem and roots, Dragendroff’s reagent was added. It showed reddish brown precipitate.

 

4. Test for tannins:

1-2 ml of 1% lead acetate was added in 2ml of test solution. A yellowish precipitate was formed which indicates the presence of tannins.

 

5. Test for steroids:

(A)    To 2-3ml test solution, 2ml of chloroform and concentrated H₂SO₄ was added sidewise. Formation of red colour in chloroform layer confirms the presence of steroids.

(B)    To 2-3ml test solution, 2ml of chloroform, concentrated H₂SO₄ and acetic acid each was poured. The presence of steroids in the extract was confirmed by formation of greenish colour.

 

6. Test for flavonoids:

To 2-3ml test solution, 5ml of dilute ammonia solution and concentrated sulphuric acid was added. The presence of flavonoids in the extract was confirmed by formation of yellow color.

 

7. Test for saponins:

5ml of test solution and 20ml of distilled water was mixed and then shaked in a graduated cylinder for 15 seconds. A persistent foam was observed which indicates the presence of saponins.

 

8. Test for phenolic compounds:

The addition of 1% ferric chloride solution to the solutions, showed formation of intense green colour.

 

9. Test for proteins and amino acids:

Test solutions with 2ml of Millon’s reagent showed white precipitate, which turns red upon gentle heating. 

 

10. Test for terpenoids:

2-3 ml cannabis extract was dissolved in 2 ml of chloroform and evaporated on water bath to dryness. To this, 2 ml of concentrated H₂SO₄ was added and heated for about 2 min. The presence of terpenoids was confirmed by grayish colour.

 

RESULTS:

Morphology of plant:

Leaf:

The leaves are dull green in colour, 9.5-10.5 cm long, and 7-9 cm in width with iconic shape. The leaves has strong odour. The leaves are palmate in shape which is divided in seven lobes.  The number and shape of lobes is not constant.

 

Stem:

Stems are thin, erect and cylindrical in shape, 13-15 cm long and 0.4-0.6cm in width. It has dark green colour.

 

Root:

Roots are cylindrical with tapering ends and white in colour. The length and width are 14-16cm and 0.4-0.6 cm respectively.

 

Microscopy of plant: 

T. S. of cannabis leaf:

 

Figure 1: T. S. of cannabis leaf

 

The microscopic study of cannabis leaf shows presence of epidermal cells as upper and lower layer of leaf. Upper epidermal layer is followed by endodermis layer. The vascular bundle stained in pink colour is present at center. It is surrounded by spongy parenchymatous cells. The upper and lower epidermis is covered by abundant covering trichomes and glandular trichomes (Figure 1).

 

Cannabis stem has outer epidermal layer covered with trichomes. Radial pattern of cortex and vascular bundles are present. Pericycle is present beneath cortex. The central region is filled with vascular bundles with parenchymatous pith region at center (Figure 2).

 

T.S. of cannabis stem:

 

Figure 2: T. S. of cannabis stem

 

T.S. of cannabis root:

 

Figure 3: T. S. of cannabis root

 

The transvers section of root shows outermost epiblema layer. It is followed by wide region of cortex. Endodermis cell layer is present below cortex. Endodermis layer has pericylic layer at its lower region. Pith is central region of the cannabis stem surrounded by vascular bundles (Figure 3).

 

Phytochemical analysis of Cannabis sativa Linn:

Results obtained for the phytochemical screening of phytochemicals in C. sativa leaf, stem and roots are given in Table 1.

 

Table 1: Preliminary phytochemical screening of C. sativa

 

DISCUSSION:

Cannabis sativa is three or four feet tall and densely branched. The preliminary phytochemical screening of leaf, roots and stems of Cannabis sativa showed that the carbohydrates and saponins are absent. The leaf, stem and root macroscopy and microscopy has been discussed. It confirms the presence of alkaloids, tannins, phenolic compounds, proteins, amino acids, flavonoids and steroids in the plant.

 

The wild grown cannabis plant which was collected nearby badhal thore was matched with the cultivated plant macroscopically, microscopically and phytochemically.

 

CONFLICT OF INTEREST:

The author declares no conflict of interest.

 

REFERENCES:

1.      Hartsel JA et al. Cannabis sativa and hemp. In Eslevier’s. Nutraceuticals: efficacy, safety and toxicity. Edited by Gupta R. 2016; 1^st ed: pp. 735-760.

2.      McPartland JM et al. Hemp diseases and pests: management and biological control - an advanced treatise. CABI publishing, Wallingford, United Kingdom. 2000.

3.      Britannica, The editors of encyclopaedia. "Cannabis". Encyclopedia Britannica, 7 Jan. 2021, Accessed 21 August 2021 available on https://www.britannica.com/plant/cannabis-plant.

4.      Russo E. Cannabis in India: Ancient lore and modern medicine. Cannabinoids as Therapeutics. Edited by Mechoulam R. Birkhauser Verlag/Switzerland. 2005. pp. 1 –22.

5.      An introduction to Cannabis sativa. Drug Times. Accessed 21 August 2021 available on https://www.drugtimes.org/marijuana/an-introduction-to-cannabis-sativa.html

6.      Atakan Z. Cannabis, a complex plant: different compounds and different effects on individuals. Therapeutic Advances in Psychopharmacology. 2012 December: pp. 241-254. doi:10.1177/2045125312457586

7.      O'Brien M. Cannabinoids. Encyclopedia Britannica. 9 October 2019. Accessed 01 August 2021 available on https://www.britannica.com/science/cannabinoid.

8.      Koppel BS et al. Systematic review: Efficacy and safety of medical marijuana in selected neurologic disorders. Report of the Guideline Development Subcommittee of the American Academy of Neurology. Neurology. 2014. 82: 1556-63.

9.      Russo EB. History of cannabis and its preparations in saga, science, and sobriquet. Chem Biodivers. 2007 August. 4(8): pp. 1614-1648. doi: 10.1002/cbdv.200790144.

10.   Turner CE et al. Constituents of Cannabis sativa L. XVII. A review of the natural constituents. J Nat Prod. 1980 March-April; 43(2): pp. 169-234. doi: 10.1021/np50008a001.

11.   Maroon J and Bost J. Review of the neurological benefits of phytocannabinoids. Surg Neurol Int. 2018 April 26. 9:91. doi: 10.4103/sni.sni_45_18.

12.   Khandelwal KR. Practical pharmacognosy Experiments and Techniques. Nirali Prakshan. 19^th ed.  2008. pp. 138 – 149.

 

 

Received on 31.08.2021         Modified on 19.11.2021

Accepted on 30.12.2021       ©A&V Publications All right reserved