South African Journal of Botany 174 (2024) 876
886

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Cytotoxic potential of Hemp seed oil and molecular docking studies with
inflammatory markers of diabetic cardiomyopathy
Rohit Chauhana, Keshav Agarwalb, Kumud Balac, Anju Krishnand, Swagata Tavharee,
Davide De Rossif, Alessio Fabbrog, Yash Sharmah,*
a
Department of Research and Development, Incare Lab, Himachal Pradesh, India
b
Cannarma Private Limited, Delhi, India
c
Therapeutics and Molecular Diagnostic Lab, Amity Institute of Biotechnology, Amity University, Uttar Pradesh, India
d
Department of Chemistry, Sathyabama Institute of Science and Technology, Kamaraj Nagar, Semmancheri, Chennai 600119, Tamil Nadu, India
e
Dr. D Y Patil College of Ayurved & Research Centre, Pimpri, Pune, Maharashtra, India
f
Ambra Srl, Siena, Italy
g
Genomics and Biotechnology, Universitat Autonoma de Barcelona, Spain
h
Department of Biotechnology, IILM University, Greater Noida, Uttar Pradesh, India

A R T I C L E I N F O A B S T R A C T

Article History: Present study was to assess the potential cytotoxic effects of commercially available Hemp seed oil and the
Received 17 April 2024 effectiveness of its compounds in inhibiting inflammatory markers associated with diabetic cardiomyopathy.
Revised 29 July 2024 The bioactive components of Hemp seed oil were analysed using gas chromatography coupled with mass
Accepted 20 September 2024
spectrometry (GC
MS). To evaluate the oil’s cytotoxic properties, a modified 3-(4, 5-dimethylthiazol-2-yl)-2,
Available online xxx
5-diphenyltetrazolium (MTT) assay was conducted on L929 cells (a mouse fibroblast cell line). Furthermore,
Edited by: Dr A. Andrade-Cetto an in vivo antiangiogenic assessment of the oil was performed using the chick chorioallantoic membrane
(CAM) model, following a laboratory-standardized protocol. Docking and simulation studies were carried out
Keywords:
to examine the binding and stability of the oil’s compounds with inflammatory markers relevant to diabetic
Hemp seed
cardiomyopathy. The GC
MS analysis revealed that hemp seed oil contains several compounds, including
Oil
Cannabidiol, Benzenemethanaminium, Dronabinol, Cannabinol, and Squalene. The cytotoxicity assessment
CAM model
Antiangiogenic on L929 cells demonstrated that Hemp seed oil did not exhibit any harmful effects at a concentration of 2.5 -
Simulation 5 mg/mL. The CAM model confirmed the presence of antiangiogenic properties in Hemp seed oil, as evi-
Diabetic cardiomyopathy denced by the inhibition of vascular patterns and blood vessel dissolution in the eggs. Additionally, docking
and molecular dynamics (MD) simulations indicated that Benzenemethanaminium and Cannabielsoin exhib-
ited the highest inhibitory efficacy and stability when interacting with IL-1b and C-reactive protein, respec-
tively. This study suggests that Hemp seed oil possesses anti-inflammatory and anti-angiogenic properties,
making it a potential herbal remedy for patients with diabetic cardiomyopathy.
© 2024 SAAB. Published by Elsevier B.V. All rights are reserved, including those for text and data mining, AI
training, and similar technologies.

1. Introduction hormones and cytokines induced by chronic hyperglycemia (Jia et al.,

2018). Heart failure and related morbidity and mortality are increasing
Diabetes mellitus (DM) and cardiovascular disease were shown to at an alarming rate, in large part, because of increases in aging, obesity,
be linked; in particular, it has been observed that increase in diabetic and diabetes mellitus. The clinical outcomes associated with heart fail-
mellitus (DM) can lead to major risk for heart failure, including hyper- ure are considerably worse for patients with diabetes mellitus rather
tension coronary heart disease and valvular heart disease (Murtaza et than those who do not present this condition (Jia et al., 2018). Type 2
al., 2019). DM has caused increased and relative development in left diabetes (T2D) has a pandemic level of prevalence. Insulin resistance
ventricular wall thickness and mass. Pathophysiology of Diabetic car- and decreased insulin secretion are traits of T2D. Inflammation has
diomyopathy (DCM) involves complex metabolic pathways such as become a significant factor in the pathophysiology of T2D, with pro-
alteration in fatty acid metabolism and changes in circulating inflammatory mechanisms damaging the pancreatic islets as a result.
Numerous cytokines and chemokines work in conjunction with each
other i.e. tumour necrosis factor-alpha (TNF-a), interleukin (IL)-6, IL-8,
* Corresponding author at: Department of Biotechnology, IILM University, Greater
IL-1b and C-reactive protein (CRP) that are considered as inflamma-
Noida, Uttar Pradesh, India.
E-mail address: [email protected] (Y. Sharma).
tory markers (Ramesh et al., 2022). Due to their accessibility and

https://doi.org/10.1016/j.sajb.2024.09.052
0254-6299/© 2024 SAAB. Published by Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.
R. Chauhan, K. Agarwal, K. Bala et al. South African Journal of Botany 174 (2024) 876
886

important traditional history, there is an increasing worldwide trend 2.2. Cell lines
to employ medicinal plants as preventive and therapeutic agents to
manage diabetes and its long-term consequences, such as cardiovascu- L929 (Mouse fibroblast cell line) were purchased from the
lar problems (Shabab et al., 2021). Several metabolites present in dif- National Centre for Cell Sciences, Pune, India. The cells were main-
ferent plants have shown the ability to inhibit the signalling pathways tained at 37 °C in DMEM medium supplemented with 10 % FBS, strep-
mediating the neurological and cancerous diseases. tomycin (100 mg/mL) and penicillin (100 U/mL), in a humidified
Hemp seeds are obtained from the specie Cannabis indica which is atmosphere of 50 mg/mL CO2.
a flowering plant belonging to the family of Cannabaceae. It generally
originated from eastern and central Asia and its major important
2.3. GC
MS profiling
traits include internodal length, leaf size and structure. C. Indica
plants tend to grow shorter and bushier than the tall and leggy C. sat-
(Al-Nemari et al., 2020).
iva plants (Karki and Rangaswamy, 2023). For generations, India’s
The bioactive compounds of the hemp seed oil were investigated
native variety of Cannabis indica has been growing unrestrictedly
using gas chromatography coupled with mass spectrometry
along the slopes of the Himalayas and the nearby plains. Hindus, who
(GC
MS). For GC
MS analysis, samples were submitted to the cen-
are primarily from India and Nepal, consume Cannabis on a regular
tral instrumentation laboratory at CUPB Ghudda, Bathinda. The anal-
basis, and it has been depicted in numerous Hindu scriptures (Mold,
ysis was performed on a Shimadzu GCMS-QP-2010 plus system with
2017). Different compounds has been isolated from Cannabis such as
a thermal desorption system TD 20. An RTx-5 Sil MS column
delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) that have
(30 m £ 0.25 mm id £ 0.25 mm film thickness) was used. The operat-
shown medicinal potential (Atakan, 2012). Previous studies also
ing conditions of the column included an oven temperature program
demonstrated that in hemp seed oil, THC concentrations ranged from
from 100 °C to 280 °C with a hold time of 1 min, and from 250 °C to
0.3 to 19.73 mg/mL, CBD concentrations ranged from 6.66 to
280 °C with a hold time of 5 min. The final temperature was main-
63.40 mg/mL, and CBN concentrations ranged from 0.11 to 2.31 mg/
tained for 20 min. The injector temperature was set at 280 °C, with a
mL. The CBN/THC ratios varied between 0.12 and 0.42, while the
sample injection volume of 0.3 mL, pressure at 76.2 kPa, total flow of
CBD/THC ratios ranged from 3.21 to 22.50. Additionally, the
50 mL/min, column flow of 1.04 mL/min, linear velocity of 37.9 cm/s,
(THC + CBN)/CBD ratios in all hemp seed oil samples were less than
purge flow of 3.0 mL/min, and a split ratio of 1.0. The ion source tem-
one (Jang et al., 2020).
perature was 230 °C, with a scan mass range of m/z 45
800 and an
Cannabis present in India has shown important medicinal proper-
interface line temperature of 280 °C. The compounds were identified
ties, our present study deals with the screening of cytotoxic potential
by comparing their mass spectra with data from NIST 11 (National
of commercially available Hemp seed oil on fibroblast cell line and its
Institute of Standards and Technology, US) (Al-Nemari et al., 2020;
inhibitory efficacy with the inflammatory markers possessing dia-
Sharma et al., 2023b).
betic cardiomyopathy.

2.4. Cytotoxic activity on cell line

2. Material and methods
Cytotoxic activity of oil was determined using a modified 3-(4, 5-
2.1. Source of sample collections dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) assay on L929
cells (Mouse fibroblast cell line) (Mosmann, 1983). Briefly, Cells were
Cannarma Ultra-Premium Multipurpose Hemp Seed Oil was pur- seeded (»5 £ 10 3 cells/well) into flat-bottomed 96-well culture
chased from Cannarma Private Limited, Delhi, India and used plates and incubated for 24 h at 37 °C using 5 % CO2 and 95 % air. 1 g/
throughout the project. mL of Hemp seed oil was dissolved in DMSO. 50 mg/ml of Hemp Seed

Fig. 1. Gas chromatography-mass spectrometry profile of Hemp seed oil.

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Table 1
The major compounds identified in Hemp seed oil: retention times (RT), Area of the peak (%), Name, Formula, molecular weight (MW) and 2D structure.

Peak# R.Time Area% Name Class of Compounds Formula MW (g/mol) 2D structure

1 3.73 0.1 1,1,3-Triethoxypropane Ester C9H20O3 176.25

2 15.062 0.34 Hexadecane Allkanes C16H34 226.44

3 20.351 0.17 Neophytadiene Sesquiterpenoids C20H38 278.5
4 21.499 9.09 Lidocaine Antiarrhythmic agent C14H22N2O 234.34

5 21.807 0.28 7,9-Di-tert-butyl-1-oxaspiro[4.5]deca-6,9-diene-2,8-dione Oxaspiro compound C17H24O3 276.4

6 22.824 7.6 Dibutyl phthalate Ester C16H22O4 278.34

7 23.589 1.03 Hexadecanoic acid, ethyl ester Fatty Acid C18H36O2 284.5

8 26.81 3.38 Linoleic acid ethyl ester Fatty Acid C20H36O2 308.5

9 26.928 2.04 Ethyl Oleate Fatty Acid Ester C20H38O2 310.5

10 27.439 0.26 Octadecanoic acid Fatty Acid C20H40O2 312.5

11 30.611 0.67 Cannabinol Phenolic Compounds C21H30O2 314.5

12 30.706 0.39 Cannabicyclol Cannabinoid C21H30O2 314.5

13 31.35 0.06 Methyltrivinylstannane Inorganic Compound C7H12Sn 214.88

14 31.456 35.85 Cannabidiol Phytocannabinoid C21H26O2 310.4

15 31.639 2.25 Silane Organometallic compounds H4Si 32.117

16 32.044 0.32 Pregnan-3-one, (5.alpha.)- Steroid Hormone C21H34O 302.5

17 32.263 0.43 Dronabinol Cannabinoid C21H30O2 314.5

18 32.412 1.6 Cannabielsoin Phytocannabinoid metabolite C21H30O3 330.5

19 32.543 16.14 Benzenemethanaminium Organic salt C29H34ClN3O2 492.0

(continued)

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Table 1 (Continued)

Peak# R.Time Area% Name Class of Compounds Formula MW (g/mol) 2D structure

20 32.699 11.25 Dronabinol Cannabinoid C21H30O2 314.5

21 33.15 0.15 Ethyl erucate Ester C24H46O2 366.6

22 33.473 3.57 Cannabinol Phenolic Compounds C21H26O2 310.4

23 34.696 0.54 Eicosane Organic compounds C20H42 282.5

24 36.234 2.49 Squalene Organic compounds C30H50 410.7

Fig. 2. (I): Images of L929 cell line in inverted light microscopy after the exposure of Hemp seed oil: untreated L929 cells (A); (B-C) L929 cells treated with 1 mg/mL of Hemp seed oil
(B); L929 cells treated with 2.5 mg/mL of Hemp seed oil (C) and Cells treated with 5 mg/mL (D). (II): Effect of Hemp seed oil on mouse fibroblast cell line L929. Morphological
changes showing proliferation of L929 Cells using specific values of Hemp seed oil after 48 hr of treatment. The cells were evaluated under an Olympus inverted microscope using
10X objective lens (magnification 100X). Scale bar: 100 mm. Untreated cells was considered negative control. The values are represented as mean § standard of three experiments.
*p < 0.05, **p < 0.01.

oil further diluted to various concentrations (0.05- 2.5 mg/mL) for cell solubilized. Finally, an automated microplate reader was used to
treatment. Total volume of 10 ml of MTT reagent (5 mg/mL) was measure the formazan’s absorbance at 570 nm (Bio-Rad, Illinois,
added after 48 h of incubation, and the mixtures were then re-incu- USA). The experiments were performed in triplicates. By comparing
bated for 3 hr. With DMSO (100 ml), the resultant formazan was the absorbance between the samples and a negative control (DMSO),
the cell viability (%) was calculated (Gul et al., 2013).
The percent inhibition was calculated by using the following for-
mula:

%Inhibition ¼ 100:::ðmean OD of test compound:::mean OD of negative controlÞ=

ðmean OD of positive control :::mean OD of negative controlÞ  100

2.5. Antiangiogenic activity on chick chorioallantoic membrane (CAM)

model

The chick chorioallantoic membrane (CAM) model was used to

Fig. 3. Antiangiogenic activity of Hemp seed oil on chick chorioallantoic membrane
model. Embryos were treated after 72hr with 1 % DMSO as a control (A.); 1 mg/mL of analyze in vivo antiangiogenic activity of oil as per laboratory stan-
Hemp seed oil (B.) and 2.5 mg/mL of oil (C.). Indicates inhibition of vasculature dardized protocol (Tayal et al., 2019). Oils in different concentrations
pattern, and Indicates dissolution of blood vessels. were applied using disc on the eight-day old eggs through cutting
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886

Table 2
Estimated DG (Kcal/mol) between the different compounds present in the Hemp seed oil and the inflammatory markers for dia-
betic cardiomyopathy.

Compounds C Reactive Protein IL6 (1ALU) TNF-Alpha (2AZ5) IL8 (4XDX) IL-1b (1ITB)
(1B09)

Lidocaine
6
4.9
5.1
6.1
5.8

Dibutyl phthalate
5.4
4.5
4.8
6
5.9
Linoleic acid ethyl ester
4.7
4.2
3.8
5
5.3
Ethyl Oleate
4.5
3.9
3.4
4.8
5.3
Cannabidiol
7.3
5.6
5.8
6.3
7.3
Cannabielsoin
7.7
6.3
6.4
7.1
7.2
Benzenemethanaminium
8.7
5.6
5.5
8.8
9.3
Dronabinol
7.8
7.2
6.5
7.6
7.8
Cannabinol
7.5
6.8
6.8
8.6
7.7
Squalene
6.4
6.4
4.9
6.4
6


small windows on the egg shell (under sterile condition). Windows <1.0 A were clustered together and addressed by the binding result
were sealed with tape and allowed to incubate for 72 hrs. After incu- with the lowest free energy of binding (Mollica et al., 2019). For fur-
bation, windows were reopened and determination of antiangiogenic ther investigation, the posture with the lowest binding energy or
activity was done by observing number of blood vessels and branch- affinity was excised and aligned with the receptor structure. A 2-D
ing compared to the control groups (Sharma et al., 2023c). representation of the interaction between amino acids and molecules
was analysed using Discovery Studio 2021 visualization tool (https://
2.6. Molecular docking AutoDock/Vina docking discover.3ds.com /discovery-studio-visualizer-download).

3D SDF files were retrieved from NCBI Pubchem database (https://

pubchem.ncbi.nlm.nih.gov/) for docking. From Protein data bank 2.7. Pharmacokinetics and drug-likeness studies
(https://www.rcsb.org), PDB files of C Reactive Protein (PDB id:
1B09), IL6 (PDB id: 1ALU), TNF-Alpha (PDB id: 2AZ5), IL8 (PDB id: The online tool Swiss ADME of the Swiss Institute of Bioinformat-
4XDX), IL-1b (PDB id: 1ITB) were downloaded. AutoDock Tools was ics (http://www.sib.swiss) was used to perform pharmacokinetics
used to create PDBQT files and grid boxes (ADT). and drug-likeness prediction for tannin and flavonoids, through the
ADT assigned the protein polar hydrogens and unified atom Koll- evaluation of their individual ADME behaviour (Daina and Zoete,
man charges. The grid map was created using a grid box using Auto- 2016). The purpose of the analysis was to determine whether such
Grid software. In relation to the following specific protein, the grid’s substances inhibited the activity of cytochrome P450 (CYP) family
centre was established at dimensions x, y, and z with a grid spacing isoforms, including CYP1A2 and CYP2D6. Additionally, drug-likeness
of 0.375 and a size of xyz points. AutoDock/Vina was employed for prediction techniques like the Lipinski, Ghose, and Veber rules have
docking using protein and ligand information along with grid box been used, as well as the bioavailability score and pharmacokinetics
properties in the configuration file (Trott and Olson, 2010). The (such as gastrointestinal absorption, P-glycoprotein, and blood brain
results with a positional root-mean-square deviation (RMSD) of barrier) (Ghose et al., 1999; Lipinski et al., 2001; Veber et al., 2002).

Fig. 4. Inflammatory receptors proliferating diabetic cardiomyopathy.

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Fig. 5. In silico docking of the Cannabielsoin, Benzenemethanaminium with Inflammatory markers in diabetic cardiomyopathy.

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Table 3
Pharmacokinetics and drug-likeness prediction by Swiss ADME.

Compound Pharmacokinetics Drug-Likeness

GI absorption BBB permeant P-gp CYP1A2 inhibitor CYP2D6 Inhibitor Log Kp (skin Lipinski Ghose Veber Bioavailability Score
permeation) cm/s

Lidocaine High Yes No No No
6.12 cm/s Yes Yes Yes 0.55

Dibutyl phthalate High Yes No Yes Yes
4.80 cm/s Yes Yes Yes 0.55
Linoleic acid ethyl ester High No No Yes No
2.97 cm/s Yes No No 0.55
Ethyl Oleate Low No No Yes No
2.49 cm/s Yes No No 0.55
Cannabidiol High Yes No No Yes
3.59 cm/s Yes No Yes 0.55
1,6-Dibenzofurandiol/ High Yes No No No
4.46 cm/s Yes Yes Yes 0.55
Cannabielsoin
Benzyldiethyl-(2,6- High Yes Yes Yes No
4.02 cm/s Yes No Yes 0.55
xylylcarbamoylme-
thy/Benzenemethana-
minium,
Dronabinol High Yes No No Yes
3.27 cm/s Yes No Yes 0.55
Cannabinol High Yes Yes Yes Yes
3.86 cm/s Yes No Yes 0.55
Squalene Low No No No No
0.58 cm/s Yes No No 0.55

The Lipinski, Ghose and Veber rules were applied to observe drug showing peaks of the number of compounds from oil that were rec-
likeness to predict whether a compound is likely to be a bioactive ognized by relating their peak retention time, peak area (%), height
according to some important parameters such as molecular weight, (%) and mass spectral fragmentation patterns to that of the known
Log P, number. The Swiss ADME tool used vector machine algorithm compounds described by the National Institute of Standards and
(SVM) (Oyaizu, 1968) with fastidiously cleaned large datasets of Technology (NIST) library.. The majority of them was represented by
known inhibitors/non inhibitors as well as substrates/non-substrates. Lidocaine, Dibutyl phthalate, Linoleic acid ethyl ester, Cannabidiol,
Benzenemethanaminium, Dronabinol, Cannabinol and Squalene.
2.8. Molecular dynamic simulation Their identification is described in Table 1 with the following charac-
teristics: retention time (RT), Area of the peak, molecular weight, for-
Desmond V 5.9 package (Schrodinger 2019-3) was used to simu- mula and their 2-D structures.
late a study between a ligand and protein to observe the changes
(Bowers et al., 2006; Release, 2019). The OPLS forcefield was used for
the MDS of docked complex (Cannabielsoin, Benzenemethanami- 3.2. Cytotoxic effect on fibroblast cell line
nium) (Banks et al., 2005). For dynamic simulation, the complex was
placed in the centre of an orthorhombic cubic box filled with TIP3P Toxicity potential of oil was estimated on the L929 cells using MTT
water models and buffers at a 10 distance from the box edge to the assay. Fig. 2 shows the percentage of cell viability of the cells treated
protein atom (Shivakumar et al., 2010). In order to neutralise the sys- with different concentrations (0.05
5 mg/mL) of oil after 48hr of
tem, counterions such as Na+ and Cl were randomly injected to the treatment. 100 % was considered as percentage of viability of
boundary condition box volume (Kaminski et al., 2001). The NPT untreated cells. Oil has shown 60.08§1.07 % of cell viability treated
ensemble with the temperature 300 K and a pressure 1 bar applied with 0.05 mg/ml of oil and viability of cells across the treatment sug-
for the run. The simulation length was 50 ns with a relaxation time of gests that cells treated has shown increase in viability of cells. This
1 ps for the complex. The long-range electrostatic interactions were signifies that with the increase concentration of oil failed to reveal
calculated by the particle mesh Ewald method (Ivanova et al., 2018). the cytotoxic effect on the proliferation of L929 cells and has shown
The Simulation Interaction Diagram tool from the Desmond MD significantly maximum viability of cells i.e. 99.47§0.06 % and 99.92§
package was used to examine the behaviour and interactions 0.055 % at 2.5 mg/mL and 5 mg/mL of oil concentration, respectively.
between the ligands and protein. The protein and ligand atom posi-
tions in time were tracked using the rmsd. The RMSD of the positions 3.3. Antiangiogenic activity
of the ligand and protein atoms over time was used to track the sta-
bility of MD simulations. In order to make sure the system is stable, Antiangiogenic activity of oil was observed on chick chorioallan-
energy levels were determined at the end of 50 ns (Sharma et al., toic membrane (CAM) model. Eight-day old embryos were selected
2023a). for treatment with oil and observed after 72 hr of incubation period.
It was observed that 2.5 mg of Hemp seed oil lead to maximum inhi-
2.9. Statistical analysis bition of vasculature pattern and dissolutions of blood vessels com-
pared to control, as shown in Fig. 3.
Each experiment was done in triplicates to estimate the accuracy
of the experimental data, and the result was expressed as the mean
§ standard deviation of three replications. P value < 0.05 was 3.4. Molecular docking
regarded as significant.
Docking analysis was done to determine the binding affinities of
3. Results the identified compounds with receptors of Inflammatory markers in
diabetic cardiomyopathy. It was found that Benzenemethanaminium
3.1. GC
MS profiling has shown maximum binding energies with the IL-1b and IL-8 i.e.

9.3 and
8.8 Kcal/mol, respectively. Cannabielsoin has also shown
It was done to detect the presence of bioactive compounds in the effective binding affinities with C reactive protein as per the ΔG
Hemp seed oil. It has revealed the presence of 24 compounds as (Table 2 and Fig. 4-5). Furthermore selected compounds were further
shown in Fig. 1. The chromatogram of GC
MS spectra analysis proceed for ADME screening and simulation studies.
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886

Fig. 6. Analysis of RMSD trajectories for complexes formed with amino acids of the C reactive protein throughout 50 ns all atom MD Simulation. (A) Interaction of Cannabielsoin
with amino acids of C Reactive Protein (1B09) (A), Showing protein information of input file (C Reactive Protein) (B), Protein
ligand root-mean-square deviation (RMSD) plot of
Rutin bound to the inhibitory site of C Reactive Protein (C), Docked complex before and after MD simulation respectively (D-E) and Histogram of the protein
ligand complex
between Cannabielsoin and C Reactive Protein (F).

3.5. Screening for pharmacokinetics and drug-likeness shown to be inhibitors of CYP1A2 and CYP2D6. Additionally, a drug-
likeness prediction was carried out based on the chosen Lipinski,
By subjecting compounds to in silico ADMET screening using the Ghose, and Veber guidelines and bioavailability score, all of which
DS 2.5 program, pharmacokinetics and drug likeness investigations are listed in Table 3.
were also carried out. Molecular docking with receptors and bioactive
components of hemp seed oil has showed inhibitory activity. Accord- 3.6. MD simulation studies
ing to study it was found that Lidocaine, Dibutyl phthalate, Linoleic
acid ethyl ester, Cannabidiol, Cannabielsoin, Benzenemethanami- Highest docked complexes were further developed for the MD
nium, Dronabinol, Cannabinol have shown highest absorption and simulation to check its stability. From the docking analysis it was
BBB permeability. Only Dibutyl phthalate and Cannabinol have observed particular binding energy between Cannabielsoin and the
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Fig. 7. Analysis of RMSD trajectories for complexes formed with amino acids IL-1b throughout 50 ns all atom MD Simulation. Interaction of Benzenemethanaminium with amino
acids of IL-1b (1ITB)(A), Showing protein information of input file (IL-1b) (B), Protein
ligand root-mean-square deviation (RMSD) plot of Rutin bound to the inhibitory site of IL-1b
(C), Docked complex before and after MD simulation respectively (D-E) and histogram of the protein
ligand complex between Benzenemethanaminium and IL-1b.

amino acids of C Reactive Protein (Fig. 6A and Fig. 6B), and the inter- involved in protein
ligand simulations (Fig. 6E). As far as the histo-
action between Benzenemethanaminium and amino acids of IL-1b gram was concerned, Phe 39, Trp 67, Val 86 and Val 94 have shown
(1ITB) has shown maximum ΔG (Fig. 7A and 7B), which was explored the hydrophobic interaction and Pro87, Glu88 have shown H-bond
for the possible atomic details in the solvent system. interaction with Cannabielsoin (Fig. 6F).
Moreover, from MD Simulation studies, it was found that C Reac- IL-1b protein structure was found conserved with a fluctuation of
tive protein was conserved with a maximum fluctuation of 2.8 Ǻ at 4.2 Ǻ at 30 ns and it goes up to 4.8 Ǻ at 50 ns (Fig 7C). Benzenemetha-
30
40 ns and later drop down to 2.5 Ǻ at 50 ns (Fig. 6C). Cannabiel- naminium also exhibited a conformational change reaching 7.2 Ǻ at
soin has also exhibited a conformational change going from 6.5 Ǻ at 50 ns and then drop down to 6.4 Ǻ at 50 ns (Fig 7C). His 301, Asn 204,
around 30
40 ns down to 4 Ǻ at 50 ns (Fig. 6C). Val86, Thr 90, Val 89, Leu 110, Met 148, Ser 152, Glu 265, Leu 6, Leu 237, Phe 150 exhibited
Trp 67,Val 94 exhibited a hydrogen bond with Cannabielsoin (Fig. 6 a hydrogen bond with Benzenemethanaminium (Fig. 7D), whereas
(D)), whereas Trp67 has shown significant amino acid interaction Lys270, Glu 202, Gln 149 have shown significant amino acid
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R. Chauhan, K. Agarwal, K. Bala et al. South African Journal of Botany 174 (2024) 876
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interaction involved in simulation (Fig. 7E). Moreover, in the histo- and anti-angiogenic agent that can be utilized as herbal remedy for
gram, Leu 6, Cys 8, Met 44, Phe 150 and Lys 270 showed hydrophobic the treatment of diabetic cardiomyopathy patients.
interaction, whereas Glu 149 revealed the H-bond interaction with
Benzenemethanaminium (Fig. 7F). MD simulation studies showed us Declaration of competing interest
that C Reactive Protein and IL-1b possess good degree of interactions
with Benzenemethanaminium and Cannabielsoin. The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
4. Discussion ence the work reported in this paper.

Cannabis is mentioned in many available Ayurvedic Granthas

(Aborginal Texts/Compendium) popularly known as Samhitas, Chi- CRediT authorship contribution statement
kitsa Grantha, Rasa Granthas and Nighantus (Understanding the
Therapeutic Potential of Cannabis, n.d.). Ayurveda advocates the judi- Rohit Chauhan: Writing
review & editing, Project administra-
cious use of Vijaya after proper Shodhana (Processing). In Ayurveda tion, Investigation, Formal analysis, Conceptualization. Keshav Agar-
Bhanga is used as an ingredient of 191 formulations which is effective wal: Writing
review & editing, Visualization, Project
in various disease conditions viz. Grahani (Malabsorption syndrome), administration, Investigation, Formal analysis. Kumud Bala: Writing
Jvara (Fever), Atisara (Diarrhoea), Agnimandya (Diminished digestive
review & editing, Software, Methodology, Conceptualization. Anju
fire), Ajeerna (Dyspepsia), Prameha (Urinary disorders), Sangrhani Krishnan: Writing
review & editing, Validation, Resources, Investi-
(Irritable bowel syndrome) and many more. Hemp seed oil is known gation. Swagata Tavhare: Writing
review & editing, Visualization,
for its nutritional properties as well as for the health benefits Software, Methodology, Conceptualization. Davide De Rossi: Writing
(Mikulcova  et al., 2017). The present study deals with identification
review & editing, Investigation, Formal analysis, Conceptualization.
of bioactive compounds and cytotoxic potential of Hemp seed oil on Alessio Fabbro: Writing
review & editing, Project administration,
Fibroblast cell line (L929). From the GC
MS analysis, the identified Formal analysis. Yash Sharma: Writing
original draft, Supervision,
compounds i.e., Cannabidiol, Dronabinol, Cannabinol were found to Methodology, Formal analysis, Conceptualization.
be consistent with the expected composition of Hemp seed oil (Hsu
et al., 2021). These compounds have gained significant attention for References
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