Comparative evaluation of physicochemical, qualitative and chromatographic profile of unprocessed and processed Cannabis sativa L (Bhanga) leaves

Swagata Dilip Tavhare^1*, Rabinarayan Acharya², Bhagyashri Vaghora³, Vinay Shukla⁴

¹Associate Professor, Department of Dravyaguna, Dr. D. Y. Patil College of Ayurved and Research Center,

D.Y. Patil Vidyapeeth (Deemed to be University), Pimpri, Pune, Maharashtra-411018.

²Director General, Central Council for Research in Ayurvedic Sciences (CCRAS),

Ministry of AYUSH, Government of India, New Delhi - 110058.

³Pharmacist, Government Ayurved Hospital, Junagadh, Gujarat- 362310.

⁴Head, Pharmaceutical Chemistry Laboratory,

Institute of Teaching and Research in Ayurveda Jamnagar, Gujarat-361008.

*Corresponding Author E-mail: drswagata32@gmail.com

ABSTRACT:

Background: Cannabis (Bhanga) has been extensively explored in research for therapeutic benefits. Out of 80+ phyto-cannabinoid detected till date; out of which Tetrahydrocannabinol (THC), Cannabidiol (CBD) and Cannabinol (CBN) are therapeutically important. Psychotropic concern about cannabis is a majorly attributed to THC presence. For reducing intoxicant effect, Ayurveda recommends processing (Shodhana) of cannabis. Objectives: To evaluate impact of cannabis leaves processing techniques through pharmacognosy, photochemistry and chromatography. Material and methods: Four different processing methods; namely water-immersion (Jala-nimajjana) (JN), water-wash (Jala-prakshalana) (JP), boiling in cow’s milk (Godugdha-Swedana) (GS) and boiling in decoction of Acacia arabica (Babbul twak Kwatha-Swedana) (BKS) were performed as mentioned in Ayurveda classics. Water and ethanol extract of respective sample and processing media were prepared. Processed samples were evaluated for physicochemical, qualitative and chromatographic studies (HPTLC) and comparative evaluation was done against markers THC, CBD and CBN. Results: Processed samples showed variation in physico-chemical parameters. Functional groups like carbohydrates, phenols and steroids retained after processing in all samples. Additionally; protein and tannin groups were found in samples processed by GS and BKS method. Processing of cannabis reduced THC, CBD and CBN from samples and which transferred to media as quantified through HPTLC. In GS method, no maker visualized through HPTLC. This may be due to changes in nature of molecules caused by boiling. Reduction of THC found highest in JP (31%) followed by JN (18%) and BKS (16%) method. Conclusion: Processing showed changes in physicochemical parameters of cannabis leaves and reduces its THC content;thus; reducing its intoxicant effect without altering presence of functional group.

KEYWORDS: Bhanga, cannabis, Cannabidiol, Cannabinol, Shodhana, Processing, Tetra-hydrocannabinol

INTRODUCTION:

Cannabis sativa L. known in Ayurveda as Bhanga or Vijayais mentioned under semi-poisonous drugs (Upavisha) category of drug which has been included in Schedule E-1 of Drugs and Cosmetics Rule, 1945^1,2For its therapeutic administration; Ayurveda classics advocates processing (shodhana) techniques. Side effects like excitement, intoxication delusion, drowsiness, excessive talks, hallucinations and state of euphoria etc. are mentioned by Ayurveda and current sciences as well.³ For internal administration of cannabis or cabnnabis based product, in order to reduce intoxicant (madakari) effects, Ayurveda have advised processing (shodhana)of cannabis with various methods.^4,5

MATERIAL AND METHODOLOGY:

Collection of the test drug: Bhanga leaves were procured from Haridwar, Uttarakhanda. Pharmacognosy of the leaves was done to confirm the species of obtained sample. Impurities like stone particles, pebbles etc. were separated out manually.

Collection of media: Potable water filtered with R.O. system of the institute was used. Cow’s milk was procured from local milk supplier. Cow’s ghee was used of Govardhana brand.

Test drug specification: Sample’s nomenclature was termed by process name followed by amount of sample processed. For test purpose sample of 25 g quantity was used and nomenclature was done as JN, JP, GS, BKS for Jala-nimajjana, Jalaprakshalana, Godugdha Swedana and Babbul-twak kwatha Swedana respectively.⁶

Procedure of Jalanimajjana (water immersion) method:

Leaves of Cannabis (25 g) were tied in a muslin cloth anddipped in water overnight for 12 h. In the morning, it was squeezed until the whole soaked water was removed.Leaves were dried under the sun. After drying, it was roasted with cow’s ghee again shade dried. (Figure 1)

Procedure of Jalaprakshalana (water-wash) method: Leaves of cannabis (25 g) were tied in a muslin clothin the form of pottali (cloths tied with the material) and washed in water, and pottali was squeezed, which oozes out greenish water. The washing continues till clear water oozes after squeezing pottali. Leaves were then shade dried. (Figure2)

Figure 1: Processing of cannabis leaves by Jala-Nimajjana (water dipping) Method

Figure 2: Processing of cannabis Leaves by Jalaprakshalana (water wash) Method

Procedure of Godugdha Swedana (boiling in cow’s milk) method: Leaves of cannabis (25 g) were tied in muslin cloth’spottali, which was dipped cow’s milk (1000 mL)for boiling for three hours at 100 °C. Leaves were separated anddried. After drying, it was roasted in cow’s ghee and then shade dried. (Figure 3)

Figure 3: Proxessing of cannabis leaves by Godugdha swedana method (boiling with cow’s milk)

Procedure of Babbul-twak Kwatha Swedana (boiling in the decoction of Acacia arabica stem bark) method: Leaves of cannabis (25 g) were tied in the muslin clothand boiled in the decoction of Babbula-twak (Acacia arabica willd bark) for 12.5 min at 100 °C. Afterwards, it was dried under the sun and then triturated with cow’smilk (in sufficient quantity, which will soak the powder)and later shade dried. (Figure 4)

All processed cannabis leaves samples were powdered in grinder, and fine powder was preparedby passing through mesh no. 70. The samples were kept in an airtight container and designated accordingly.

Figure 4: Processing of cannabis leaves by Babbul twaka kwatha Swedana (Boiling with decoction of Acacia arabica stembark) method

Pharmaceutical study:

i. Physicochemical analysis:

Physicochemical analysis like loss on drying, total ash, acid-insoluble ash, alcohol and water-soluble extractive values, methanol soluble extractive value etc. were studied as per API standard.⁷

ii. Qualitative test:

The aqueous and methanol extract of unprocessed and processed sample were tested for presence of various constituents like proteins, amino acids, steroids, flavonoids, alkaloids, phenolic acid and tannin by standard procedures of practical pharmacognosy..⁸

iii. HPTLC:

Procurement of standards, plant samples and chemicals: Primary standard of THC, CBN and CBD were procured from Sigma Aldrich company. All the other chemicals and solvents used in study were of HPTLC grade (E. Merck, Mumbai, India).

Preparation of samples and standards for HPTLC:

Extracts of unprocessed sample, processed samples (JN25, JP25, GS25, BKS25) and standards THC, CBN and CBD were prepared with concentration of 10mg of sample in 1 ml of methanol. Ultra sonication of mixture was done to ensure complete dissolution.

Preparation of Fast Blue-B salt solution:

50 mg of Fast Blue-B salt powder was dissolved in 1 ml distilled water. Mixed thoroughly, added 20 ml of methanol in it, and stirred properly. A light brownish color solution was developed.

Visualization of constituents:

Developed plates of test samples were visualized by spraying Fast Blue B solution. After spraying with Fast Blue B solution scarlet, violet and pink colour gets visualized for THC, CBD and CBN respectively. By matching color resemblance with marker component, Rf value of THC, CBD and CBN standards was found as 0.83, 0.85 and 0.84 respectively. Area occupied by THC, CBN and CBD at respective Rf was calculated manually from chromatographic graph by considering the maximum peaks.

RESULTS:

Physicochemical study:

The parameters like pH, moisture content, LOD, ash value, water and methanol extractive values and acid insoluble ash of unprocessed and JN, JP, G and BK processed samples are depicted in table 1.

Qualitative test: The comparative analysis of qualitative tests performed for methanol and water extract of unprocessed and processed cannabis samples is illustrated in table 2.

HPTLC:

On HPTLC plate, THC, CBD and CBN markers were visualized in unprocessed and processed samples of cannabis. (Figure 5)

Table 1: Physicochemical analysis of unprocessed and processed cannabissamples

Sample	Foreign Matter	pH	Moisture content	LOD	Ash Value (%)	WEx %	Mex %	A.I.A. %
UP	Nil	5 @ 31	10	4.5	18.48	16.4	24.8	6.4
JN25	Nil	5.66@ 31.3	11.2	4.1	18.20	6.5	24.7	4.7
JP25	Nil	6.83 @ 31.6	8.6	6.98	17.05	7.9	9.1	2.34
GS25	Nil	5.99 @31.9	13.4	6.37	18.47	15.4	36.4	2.46
BKS25	Nil	5.97 @ 31.5	8.2	5.25	18.57	12.9	24.1	6.84

Wex= Water extract, Mex= Methanol extract, AIA= Acid insoluble ash

Table 2: Qualitative test for various functional groups inmethanol extract of unprocessed and processed cannabis sample

Sample

Carbohydrate

Protein

Amino acid

Steroid

Flavonoids

Alkaloid

Phenolic acid

Tannin

M
Ex

WEx

M
Ex

WEx

M
Ex

WEx

MEx

W
Ex

M
Ex

WEx

Mex

WEx

Mex

W
Ex

Mex

WEx

BKS

HPTLC Tracks of samples: 1.THC 2.5ug/ml, 2. THC 5ug/ml, 3. THC 7.5ug/ml, 4. CBN 7.5ug/ml, 5. CBN 10 ug/ml, 6. CBN 12.5 ug/ml, 7. CBD 2.5ug/ml, 8.CBD5 ug/ml, 9.CBD 7.5 ug/ml, 10. UP, 11. JN25 extract10 ug/ml, 12. JP25 extract10 ug/ml, 13. G25 extract10 ug/ml,14. BK25 extract10 ug/ml (After dipping in Fast blue solution)

The Rf at which maximum area was covered by respective constituents was taken into consideration. Percentage availability of phytoconstituents in processed samples and media have been given calculated by formula

% area of phytoconstituents= Area (phytoconstituent) at specific Rf in unprocessed sample- Area (phytoconstituent) at specific Rf in processed samples/Area (phytoconstituent) at specific Rf in unprocessed sample * 100

Table 3: Percentage of Tetrahydrocannabinol, Cannabidiol and Cannabinol in processed sample

Method	% THC detected in sample	% CBD detected in sample	% CBN detected in sample
JN 25	81.74%	99.53%	81.36%
JP 25	69.34%	80.45%	77.56%
GS 25	Couldn’t trace	Couldn’t trace	Couldn’t trace
BKS 25	84.12%	92.83%	89.82%

Rf for THC: 0.83, Rf for CBD: 0.85, Rf for CBN: 0.83

The extracts of media(i.e. water, cow’s milk and Acacia arabica) in which cannabis leaves were processed werevisualized through HPTLC for the presence of marker phytoconstituents THC, CBD and CBN. (Figure 6)

HPTLC Tracks of media:

1.THC 2.5ug/ml, 2. THC 5ug/ml, 3. THC 7.5ug/ml, 4. CBN 7.5ug/ml, 5. CBN 10 ug/ml, 6. CBN 12.5 ug/ml, 7. CBD 2.5ug/ml, 8.CBD5 ug/ml, 9.CBD 7.5 ug/ml, 10. UP, 11. JN25 extract10 ug/ml, 12. JP25 extract10 ug/ml, 13. G25 extract10 ug/ml, 14.BK25 extract10 ug/ml (After dipping in Fast blue solution)

DISCUSSION:

Analytical methods are one of the best techniques for detection and quantification of various cannabinoids of cannabis.⁹Processing of cannabis causes physical and chemical changes. pH value for UP sample is 5. For JN, JP, GS and BKS samples; pH ranges from 5.66@ 31.3, 6.83, 5.99 @31.9 @ 31.6 and 5.97 @ 31.5 respectively. After processing; pH gets increased in samples which mean acidic nature of cannabis is reduced. Cannabis is quoted as Pittala (increases acidity). On administration, it shows severe hyperacidity in Pitta predominant person (person sensitive to acidic food) and mild to moderate acidity among others. By processing methods recommended by Ayurveda, hyperacidity side effect can be minimized. LOD of unprocessed sample is 4.5. After processing; LOD values of the samples were found 4.1, 4.98, 6.37 and 5.25 respectively for JN, JP, GS and BKS methods. Increased LOD value in sample GS and BKS is due to addition of lipid nature particles of cow’s milk used for boiling and trituration respectively which have created stickiness and moisture in samples.The moisture content of UP sample was 10% while JN, JP, GS and BKS samples it was 11.2%, 8.6%, 13.4% and 8.2% respectively. Moisture content of drugs should be at minimal level to discourage the growth of bacteria, yeast or fungi during storage. To avoid exposure of drug to moisture; it should be kept in airtight container. Ash value ranges from 17 to 18.5% for all methods samples. The average ash value of UP, JN, JP, GS and BKS samples were 18.20%, 17.05%, 18.47% and 18.57% respectively.Water extractive value of JN, JP and BK methods were lesser than unprocessed sample.In GS method, no much changes found in water extractive value.Acid insoluble ash was found reduced in comparison to unprocessed sample while in GS method it was slightly raised probably due to weakly acidic nature of tannin. (Table 1)

Qualitative test:

Presence of carbohydrates and steroids were found in methanol and water extract of unprocessed and processed samples.¹⁰Flavonoids detected in unprocessed and all processed samples except water extract of BKS method. Proteins were detected in methanol and water extract of GS and BKS samples due to use of cow’s milk was done in processing method. Amino acids and phenolic acid were not detected in any cannabis leaves samples. Tannins were observed in BKS sample method as media used i.e. acacia bark is rich source of tannins. (Table 2) The anti-oxidant property of cannabis is due to presence of functional groups like phenols, flavonoids, diterpenes, triterpenes, steroids etc.¹¹

HPTLC:

The availability of phytoconstituents in processed sample has been calculated in comparison to unprocessed ones. There are various methods of quantification of cannabinoids.¹²After processing there is decrease in THC, CBD and CBN in samples of JN, JP and BKS methods.In GS method, no constituents visualized on HPTLC plate. This may be due continuous heat provided during processing which have caused change of phytoconstituent’s form. (Figure 5)

Average of 18%, 31% and16% THC reduction was noted in JN, JP and BKS methods samples.
It either have lost or transformed to other molecule during processing. Average 99.53%, 80% and 93% CBD was detected in JN, JP and BKS processed samples. Average 81, 78 and 90% CBN is detected in JN, JP and BKS processed samples. (Table 3, Graph 1) The availability of markers in HPTLC media plate shows removal of THC, CBD and CBN in respective media after processing. (Figure 6)

Graph 1: Percentage of THC, CBD and CBN in cannabis processed samples in comparison with unprocessed cannabis

In GS method, THC, CBD and CBN could not be traced through fingerprint technique, may be due to continuous boiling of drug with cow’s milk which might have transformed the form of THC, CBD and CBN into other chemical moieties which couldn’t visualized at particular Rf. (Figure 5 and 6)

Cannabis potency is mainly evaluated by THC, which is primary psychoactive cannabinoid in cannabis. It is reported that, all healthcare associated people are not aware of side effects of cannabis.¹³Adverse effects caused due to acute or regular use of cannabis depends upon its THC concentrations. Processing technique showed removal of 18%, 31% and 16% THC from samples JN, JP and BKS respectively. Reduction of THC by processing of leaves definitely will be helpful to reduce its side effects. Complete removal of THC is non-worthy; as it is therapeutically useful and reported for its myriad of pharmacological effects in animals as well as human.^14,15Recent research have proved evidences that cannabinoid coupled with hydroxyl-citric acid is useful for the treatment of melanoma.¹⁶Ayurveda have described various formulations of cannabis for treating health conditions like pain, irritable bowel syndrome, appetite loss, insomnia, sexual problems like loss of libido, erectile dysfunction etc.^17,18THC and CBD are the most prevalent cannabinoids in the cannabis plant which acts through endocannabinoid system.¹⁹These two cannabinoids are traditionally extracted from the plant and have been used for many different purposes like recreation, medicine, nutraceutics and cosmeceutics.

THC is main psychoactive constituent of cannabis while CBD lacks psycho activity.²⁰CBD is reported to potentiates some therapeutic effects of THC,while counteracts some of its undesirable effects like sedation, psychotropic effects, tachycardia etc.²¹CBN; after exposure to air, light and/or heat, degrades to THC. However it is non-psychoactive. This makes CBN an attractive target for therapeutic research.
CBN is potent analgesic, anti-inflammatory, appetite stimulant and relaxant in actions.²²

Processed cannabis leaves sample retains the phytoconstituents THC, CBD and CBN. Among four methods of processing, comparatively maximum i.e. 31% THC reduction was found in JP method. Being, psychoactive constituents, its reduction define decreased psychotropic effect. It has been well reported that shodhana process decreases the toxicity without altering therapeutic benefits.²³

CONCLUSION:

Processing (shodhana) of Cannabis sativa leaves causes variation in physicochemical parameters. In addition to unprocessed samples, protein and tannin groups were found in samples of cow’s milk boiling and Acacia arabica boiling method. Rest functional groups like carbohydrates, phenols and steroids remains unaltered after processing. Reduction and transformation of THC, CBD and CBN were noted in processed samples. All the processing methods help to reduced psychoactive components THC.Water-wash processing method causes highest THC reduction.

REFERENCES:

1. Sen V. Bhaishajya Ratnavali, Editor, Pandey Gyanendra, Chaukhamba Sanskrit Series office, Varanasi. 2005; Appendix C, First Edition 2005; pp. 529

2. Anonymous. Ministry of Health and Family Welfare (Department of Health), Government of India. Drugs and Cosmetics act 1940 with Drugs and Cosmetics Rules, 2003. Ministry of Health and Family Welfare (Department of Health). Delhi: 1945; pp. 317.

3. Mishra GS. Àyurveda Prakash. Chaukhamba Surabharati Academy, New Delhi, India. 2007; pp. 490–5.

4. Bhavamishra: Bhavprakasha Nighantu. Haritakyadi varga, Commentary by Prof. K. C. Chunekar. Edited by Late Dr. G.S. Pandey; Chaukhamba Bharati Academy, Varanasi.2006; pp.141.

5. Kichloo, A. et al. Marijuana: A systems-based primer of adverse effects associated with use and an overview of its therapeutic utility. SAGE open medicine, 2021; Mar 9; 9:20503121211000909. doi: 10.1177/20503121211000909.

6. Tavhare SD et al. Pharmacognostical specification of Cannabis sativa (Bhanga) leaves processed withvarious Ayurveda-recommended methods. J Drug Res Ayurvedic Sci. 2023;8 (4)

7. Anonymous. The Ayurvedic Pharmacopoeia of India. 1st ed. Part II, Vol. I. New Delhi: Government of India, Ministry of Health and Family Welfare; 2007. Appendix-2 (2.2.3), p. 140.

8. Kokate CK. Practical Pharmacognosy, Edn 4, Vallabh Prakashan, Delhi, 107-111, 1997.

9. Teotia AK, Kumar S. Screening, Identification and Quantitation of Cannabis. Asian J. Research Chem. 2009; 2(4): 401-403.

10. Komal Sharma, Rinu Rana, M S Ashawat. Preliminary phytochemical screening of leaves, stems and roots of wild Cannabis sativa. Research Journal of Pharmacognosy and Phytochemistry. 2022; 14(1): 19-2. doi: 10.52711/0975-4385.2022.00004

11. Kanittha Nakkliang et al. Assessment of Genetic relationship among Cannabis sativa L. in Thailand based on ISSR and their Phytoconstituents properties. Research Journal of Pharmacy and Technology 2023; 16(5): 2197-4. doi: 10.52711/0974-360X.2023.00361

12. Hangane Ronald, Ketan Patil, Astha Pandey. Detection of Cannabis sativa by various Analytical Techniques. Research Journal of Pharmacy and Technology. 2023; 16(6): 2917-0. doi: 10.52711/0974-360X.2023.00481

13. Nataliia Aliekperova et al. Pharmacists’ Opinions on the Legalization of Medical Cannabis in Ukraine. Research Journal of Pharmacy and Technology. 2023; 16(4): 1851-6. doi: 10.52711/0974-360X.2023.00303

14. National Academies of Sciences, Engineering, and Medicine; Health and Medicine Division; Board on Population Health and Public Health Practice; Committee on the Health Effects of Marijuana: An Evidence Review and Research Agenda.Washington (DC): National Academies Press (US); 2017 Jan 12.

15. Tavhare SD et al. Pharmacological Evaluation of Processed Cannabis Leaves as a Non Sedative Analgesic: The Novel Approach. Journal of Ayurveda. 2021; 15(3): 193-197. doi: 10.4103/joa.joa_330_20

16. S Narendra Kumar et al. Molecular Docking studies of THC-HCA on Cancer Receptors. Research Journal of Pharmacy and Technology. 2022; 15(7): 3195-9. doi: 10.52711/0974-360X.2022.00535

17. Tavhare, S., and Acharya, R. Bhanga as an activity potentiater in Ayurvedic classics and Indian alchemy (Rasashastra): A critical review. International Journal of Ayurvedic Medicine. 2016; 7(3). https://doi.org/10.47552/ijam.v7i3.829

18. Tavhare SD, Acharya R. Exploring the pharmaco-clinical view on Bhanga (Cannabis sativa Linn.): a classical unfamiliar portrayal. International Journal of Pharmaceutical and Biological Archives. 2017; 8(6): 59-78

19. Kaushik S. et al. Endocannabinoid activation and polycystic ovary syndrome: A systematic review. Research J. Pharm. and Tech. 2020; 13(1): 448-452. doi: 10.5958/0974-360X.2020.00087.6

20. Choudhary N et al. Phytochemical aspect of Cannabis sativa (L.). Research J. Science and Tech. 2013; 5(2): 284-288.

21. Russo E, Guy GW. A tale of two cannabinoids: the therapeutic rationale for combining tetrahydrocannabinol and cannabidiol. Med Hypotheses. 2006; 66: 234–246. doi: 10.1016/j.mehy.2005.08.026

22. Wong, H., Cairns, BE. Cannabidiol, Cannabinol and Their Combinations Act as Peripheral Analgesics in a Rat Model of Myofascial Pain. Arch. Oral Biol. 2019; 104: 33–39. doi: 10.1016/j.archoralbio.2019.05.028

23. Mishra SK et al. Shodhana attenuates cytotoxicity of methanolic extract of Semecarpus anacardium nuts. Research J. Pharm. and Tech. 2017; 10(2): 567-574. doi: 10.5958/0974-360X.2017.00113.5

Received on 01.12.2022 Modified on 08.05.2023

Research J. Pharm. and Tech 2023; 16(11):5341-5346.

DOI: 10.52711/0974-360X.2023.00865