In-silico and in-vitro studies of two cannabinoids of Cannabis sativa against...

[Full title: In-silico and in-vitro studies of two cannabinoids of Cannabis sativa against prostate cancer]

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119 Journal of Ayurvedic and Herbal Medicine 2023; 9(3): 119-124 Research Article ISSN: 2454-5023 J. Ayu. Herb. Med. 2023; 9(3): 119-124 Received: 27-05-2023 Accepted: 17-08-2023 © 2023, All rights reserved www.ayurvedjournal.com DOI: 10.31254/jahm.2023.9304 *Corresponding author: Prof. Bibhu Prasad Panda Microbial and Pharmaceutical Biotechnology Laboratory, Department of Pharmacognosy and Phytochemistry, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India Email: bppanda@jamiahamdard.ac.in In-silico and in-vitro studies of two cannabinoids of Cannabis sativa against prostate cancer Jasmeet Kaur 1 , Sanaa Ismael Abdul Jabar 1 , Asrar A. Malik 2 , Humaira Farooqi 3 , Sourab Agarwal 4 , Bibhu Prasad Panda 1 * 1 Microbial and Pharmaceutical Biotechnology Laboratory, Department of Pharmacognosy and Phytochemistry, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India 2 Department of Life Sciences, School of Basic Science and Research, Sharda University, Greater Noida, UP, India 3 Department of Biotechnology, School of Chemical and Life Sciences, Jamia Hamdard, New Delhi, India 4 Hempcann Solutions Private Limited, Gangadhar Meher Marg, Patia, Bhubaneswar, Odisha, India ABSTRACT The phytochemical profiling of different extracts revealed the presence of high concentration of CBD in 80:20 hydroalcoholic extract, and that of THC in 60:40 hydroalcoholic extract. The MTT assay of combination of CBD and THC highlighted the extracts as potent cytotoxic agents against prostate cancer (PC 3) cells, with IC 50 values of 1292 ng/ml, 953.3 ng/ml and 1134 ng/ml, respectively for HCP-CO 4, HCM-CO 1 & HCZ-CO 1. The molecular docking study revealed a good binding of androgen receptor (PDB ID, 2 am 9) with CBD and THC possessing binding affinity energy of -7.1 and -7.2 kcal/mol respectively. Based on the amino acid residual interaction of CBD and THC within the 2 am 9 receptor, THC reported additional hydrogen bonds as compared to CBD, suggesting it to be more potent antagonist in comparison to CBD. The present study highlighted the potential of CBD and THC as a therapeutic agent for treatment of prostate cancer Keywords: Prostate cancer; CBD; THC; MTT assay; Molecular docking. INTRODUCTION Globally, prostate cancer is the second most common cause of cancer related deaths. The range of occurrence of prostate cancer increases in older men. Even though the mechanism and etiology of development of prostate cancer is still unknown, there are various factors which are linked with development of disease such as lifestyle related factors, including smoking, diet; high levels of testosterone, family history and age [1] . Numerous treatment options which are currently available for prostate cancer includes radiation therapy, cryotherapy, chemotherapy, high intensity focused ultrasound, laparoscopic prostate surgery, radical prostatectomy, endocrine treatment and androgen deprivation therapy (ADT) [2] . The initial approach for management of pain in most cases is ADT, followed by systemic chemotherapy involving docetaxel and mitoxantrone. However, these treatment options might result in toxicity and is often associated with drug resistance [3] . Therefore, there is a huge need of natural compounds for treatment of various ailments including prostate cancer. Cannabis sativa have been in use since time immemorial for various purposes such as in medicine and textile industry. Currently, it is widely used as recreational drug possessing various impacts such as prophylactic, anti-asthmatic, analgesic, oxytocic, appetite stimulant, antidepressant, antiepileptic, hypnotic, antibiotic, tranquilizer, anticancer and topical anesthetic. It contains many secondary metabolites such as cannabinoids, flavonoids and terpenes [4] . The cannabinoids are further classified into three main groups: endocannabinoids, phyto-cannabinoids and synthetic cannabinoid. Although around 100 cannabinoids have been identified, the major ones are Δ 9 -tetrahydrocannabinol (THC) and cannabidiol (CBD). The major psychoactive component of cannabis sativa is THC which strongly binds to the CB 1 receptors in the central nervous system exhibiting antiemetic, euphoric and analgesic properties [5,6,7] . Various literatures have been reported which demonstrate the use of THC in treatment of prostate cancer [8,9,10] . CBD, on the other hand, has low affinity towards CB 1 receptors with no psychoactive properties, but instead binds to other physiological targets in the body. Due to the non-intoxicating properties of CBD, it is of great concern for the researchers. Till date, many literatures have highlighted the use of CBD as anticancer. De Petrocellis et al. 2013 [9] reported the use of CBD and inhibition of prostate cancer cells via induction of apoptosis. The current study reports the cytotoxic potential of combination of CBD and THC present in varied concentration in extracts following their docking studies.

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120 Journal of Ayurvedic and Herbal Medicine|July-September|2023 MATERIALS & METHODS Plant Materials The plant species Dried Cannabis ( Cannabis sativa ) leaves were sourced from Dept of Excise, Odisha and stored at Hempcann warehouse in ambient temperature till further use. (Voucher no: HCS/RM/HLHP/001). The materials (plant extract) were collected from R&D of Hempcann Solutions Pvt. Ltd., Bhubaneswar, Orissa, India. HPLC grade methanol and acetonitrile were obtained from S.D. Fine Chemical Pvt. Ltd., India. All other chemicals used in the experiment were of analytical grade. The leaves were washed, cleaned and dried at room temperature. The leaves were then pulverized and then grinded to fine powder and stored in air tight container. Quantification of THC and CBD by HPLC Dried extract of cannabis leaf (1 mg) was dissolved in 10 ml of methanol: chloroform (9:1 v/v) by sonicating for 15 min, followed by vortex mixing for 5, 10 and 15 minutes. The extract was then centrifuged and subjected to filtration using 0.45 µm filters and was stored for further analysis. A Shimadzu HPLC system equipped with quaternary pumps, a UV/VIS detector with variable wavelength, an SPD column oven and SCL system controllers was used for analysis. Chromatographic column equipped with reverse phase C 18 column (250 x 4.6 mm x 5 µm) was used for analysis. The mobile phase was selected based on assay parameters such as peak separation, shape, sensitivity , the cost and the amount of time required for analysis and consists of acetonitrile: water in the ratio of 80:20. The flow rate was maintained at 1 ml/min. Assays were performed by serial dilution method using various concentrations of standard in methanol and comparison was made with blank reference solvent at the wavelength of 220 nm. By using the equation obtained from the standard plot, the concentration of each sample was calculated based on the relationship between concentration and absorbance, and R 2 was calculated for each standard. MTT Assay MTT (3’ - (4, 5 – dimethylthiazol- 2-yl)- 2,5- diphenyl tetrazolium bromide) assay was conducted in PC 3 cells for evaluation of in vitro cytotoxic activity of CBD and THC. Prostate cancer (PC 3) cells were grown in DMEM, supplemented with 1% ampicillin and 10 % fetal bovine serum. The cells were maintained at 37 0 C in an incubator with 5% CO 2 . Subculturing of cells in 96 well culture plates was done at a density of 2.6 x 10 6 cells/ml. The cells were then treated with different dilutions of extracts. After 24 and 48 hours of incubation, MTT solution was added to each well, followed by 4 hours of incubation and addition of DMSO for terminating the reaction. The absorbance was read against 540 nm. The formation of formazan crystals is directly proportional to the number of live cells. Molecular Docking The in-silico docking of CBD and THC with the androgen receptor (PDB ID: 2 am 9) was performed using Auto dock vina version 112, Pymol version 2.5.1, Open babel version 3.1.1, Ligplot version JDK 8-U 51, Discovery Studio version 20.1 and UCSF Chimera version 1.10. The calculated binding free energies were compared with acarbose molecules. RESULTS Macroscopic, Microscopic and physio-chemical parameter study of plant identification is caried out (Table 1). Table 1: Macroscopic, microscopic, and physio-chemical parameter study of Dried Cannabis ( Cannabis sativa ) leaves Voucher Specimen No: HCS/RM/HLHP/001 Common name: Cannabis, Vijaya Genus: Cannabis Species: Sativa Family Name: Cannabaceae Sourced from: Department of excise, Govt. of Odisha Date of collection: 03.03.2020 Part of Plant: Dried leaf Macroscopic: The leaves are palmately compound with linear, lanceolate and slightly acrid leaflets, growing up to 20 cm long, pointed, narrow at base. The upper surface is dark green and rough, whereas the lower surface is pale, downy, with a strong and characteristic odor. Microscopic: On the dorsiventral surface of leaves and bracts, the upper epidermis is composed of unicellular, pointed, curved, conical trichomes with enlarged bases containing calcium carbonate cystoliths. In the mesophyll, calcium oxalate clusters are located in many cells consisting of usually one layer of palisade cells with a spongy fabric.

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121 Journal of Ayurvedic and Herbal Medicine|July-September|2023 Identification Specification Result Foreign matter NMT 2% 1.82% Total Ash NMT 15% 12.67% Alcohol-soluble extractive NLT 10% 62.34% Water-soluble extractive NLT 13% 42.30% Image of the Dried Leaf The cannabis sativa lin. leaves were extracted with hydro-alcoholic solvent. All the extracts were further subjected to quantification of CBD and THC using HPLC. The method was developed using a suitable mobile phase of acetonitrile: methanol (80:20). Repeated analyses of analytes during experimentation and storage of solutions at laboratory conditions and in refrigerator were conducted to evaluate stability of analytes in solution during analysis. The standard plot was constructed for CBD and THC. The leaf extracts were designated with THC & CBD content. Such as High CBD &THC; High CBD & Low THC; High THC & Low CBD (Table 2) The HPLC chromatograms of all the extracts with chromatograph peak is shown in Figure 1. Figure 1: The HPLC chromatograms of all the extracts HCP-CO 4 (Cannapain) (A), HCM-CO 1(Cannaflam) (B) & HCZ-CO 1 (Cannaron) (C) with chromatograph peak.

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122 Journal of Ayurvedic and Herbal Medicine|July-September|2023 Table 2: The Leaf extracts with code and brand name as supplied by Hempcann Solutions Pvt. Ltd., Bhubaneswar, Orissa, India No. Extract code Brand Name THC & CBD Content per ml Remark 1 HCP-CO 4 Cannapain 20 mg CBD & 20 mg THC High CBD & High THC 2 HCZ-CO 1 Cannaron 10 mg CBD & 20 mg THC Low CBD & High THC 3 HCM-CO 1 Cannaflam 20 mg CBD & 10 mg THC High CBD & Low THC To access the cytotoxic potential of THC and CBD, cell viability assay was performed using MTT assay. Different concentrations (50 ng/ml, 100 ng/ml, 200 ng/ml, 400 ng/ml, 800 ng/ml, 1 ug/ml, 2 ug/ml and 5 ug/ml) of samples (CBD + THC) were evaluated against PC 3 cells, compared to paclitaxel (Figure 2). Figure 2: The cytotoxic potential of HCP-CO 4, HCM-CO 1, HCZ-CO 1 & Paclitaxel Results demonstrated that HCP-CO 4, HCM-CO 1 & HCZ-CO 1 extracts had a significant in-vitro effect on human cancer cell line (PC 3) at all concentration except 2 and 5 µg/ml. Higher concentration (5 µg/ml) of extracts were found to be too concentrated. Interestingly, all the three extracts (HCP-CO 4, HCM-CO 1 AND HCZ-CO 1) exhibited stronger inhibition with an IC 50 of 1292 ng/ml, 953.3 ng/ml and 1134 ng/ml, respectively (Figure 3). This present study confirmed the effectiveness of cytotoxic activity of cannabis sativa lin. leaves. Figure 3: Cell cytotoxicity of different extracts of cannabis sativa with comparison to paclitaxel The docking study was carried out to predict potential binding of CBD and THC with human androgen receptor ligand (PDB ID: 2 am 9). The binding affinity energy of 2 am 9 with CBD and THC was found to be -7.1 and -7.2 kcal/mol respectively. The residual amino acid interaction of CBD and THC shows a difference in amino acid residues of both the compounds (Figure 4). This indicates that there is a difference in the activity of both compounds. However, binding of THC with the receptor results in additional hydrogen bonds with key amino acids residues of the androgen receptors (Table 3). This result suggests that THC is more potent antagonist of androgen receptor than CBD. The combined binding of CBD and THC with 2 am 9 is shown in supplementary material (Figure 5). The present study suggests that CBD and THC proves to be promising candidate for treatment of prostate cancer. Figure 4: Residual amino acid interaction of (a) CBD with 2 am 9; (b) THC with 2 am 9 Table 3: Residual amino acid interaction of CBD and THC with 2 am 9 Parameter Value Residual amino acid interaction CBD THC 681-Glu ** 682-Pro * 684-Val * 715-Val * 718-Trp *** 744-Leu * 748-Ala * 752-Arg * 678-Glu **** 681-Glu **** 682-Pro * 683-Gly **** 684-Val * 685-Val **** 751-Trp **** 752-Arg *

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123 Journal of Ayurvedic and Herbal Medicine|July-September|2023 763-Tyr * 808-Lys * 755-Thr **** 756-Asn **** 801-Pro **** 804-Phe **** 805-Leu * 808-Lys **** *Alkyl/Pi-alkyl Interaction; **Pi-anion; ***Pi-sigma; ****Hydrogen BondConclusions Figure 5: Combined binding pose of CBD and THC with 2 am 9 DISCUSSION Researchers have extensively studied the therapeutic effects of cannabinoids on cancer cells in recent years, as well as their potential anticancer effects. In addition, they affect tumor progression and development by interfering with the components of the endocannabinoid system. Our results demonstrate that cannapain, cannaflam, and cannaron tends to inhibit the growth of PC 3 prostate cancer cells in a synergistic manner. In a similar study, Scott et al [11] used neutral forms of cannabinoids in leukemia cells to study combinations of CBD, cannabigerol (CBG), and cannabigevarin (CBGV). The study demonstrated that CBD does not act antagonistically with other cannabinoids to reduce cell number, and that cannabinoid activity is affected by drug combinations and treatment schedules. Similarly, another study suggests that the level of each compound required to affect carfilzomib was reduced when THC and CBD were used in combination, indicating potential synergistic effects of the two cannabinoids together. There has also been evidence that multiple compounds, including cannabinoids, terpenes, and flavonoids, interact synergistically with chemotherapeutics, resulting in a reduction in dosage of each agent required to produce a therapeutic effect, decreasing the number of adverse effects patients may suffer from [12] . Hence, the results of combining low CBD and high THC proves to be promising in reducing cell viability, thereby ruling out cell multiplication. Furthermore, binding of CBD and THC with human androgen receptor was explored in this study. It was found that 2 am 9 had binding affinity energies of -7.1 and -7.2 kcal/mol for CBD and THC, respectively. As the residual amino acid interaction between CBD and THC shows a difference in amino acid residues, it indicates that they have different activity levels. Various molecular docking simulation studies have been performed in relation to Cannabis sativa L . A study confirmed the binding of eight cannabinoids having higher binding affinities to their respective protein targets for exhibiting the pharmacological effect of the disease [13] . Another study identified cannabinoids as binding partners of DLC 1 RhoGAP domain in liver cancer using molecular docking calculations. The calculations indicate that cannabichromene and cannabidiolic acid have a high affinity for binding to GTPaseactivating proteins. As a result of non-covalent interactions involving important amino acids, these compounds remain within the active site through hydrogen bonds, for example [14] . Although CBD has been the subject of several invitro and in-silico studies, further studies are urgently necessary to gain a complete understanding of its functions. The anticancer properties of cannapain, cannaflam, and cannron have been shown to be attributed to their ability to shrink prostate cancer cells. Aside from THC and CBD, there are additional compounds in total cannabis extracts that must also be identified and analyzed to determine their effectiveness as anti-tumor agents. Further studies are needed to determine which cannabinoid compounds are more effective and in what combination. To quantify these results, more controlled and longer-term in vitro and in vivo studies are necessary. The combined effect of CBD and THC inhibits the prostate cancer (PC 3) cells with IC 50 value of 1292, 953.3 and 1134 ng/ml respectively for HCP-CO 4, HC-C 01 and HCZ-CO 1). The molecular docking study revealed that CBD and THC are best fitted into the receptor and shows a good binding affinity to the receptor. The present study may offer important insight for designing a therapeutic dosage for treatment of prostate cancer. Furthermore, toxicity study will be carried out to find the toxic profile of the extracts. Conflict of interest statement There is no conflict of interest in publishing the research work. Acknowledgement All the author acknowledges the financial support and research materials received form Hempcann Solutions Private Limited , Gangadhar Meher Marg, Patia, Bhubaneswar, Odisha, India. ORCID ID Jasmeet Kaur: https://orcid.org/0000-0002-3438-2882 Sanaa Ismael Abdul Jabar: https://orcid.org/0000-0002-4936-3289 Asrar A. Malik: https://orcid.org/0000-0003-1950-8747 Humaira Farooqi: https://orcid.org/0000-0002-8222-7705 Sourab Agarwal: https://orcid.org/0009-0003-0884-5585 Bibhu Prasad Panda: https://orcid.org/0000-0001-5110-2945 REFERENCES 1 Ramos JA, Bianco FJ. The role of cannabinoids in prostate cancer: Basic science perspective and potential clinical applications. Indian Journal of Urology: IJU: Journal of the Urological Society of India. 2012 Jan;28(1):9.

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124 Journal of Ayurvedic and Herbal Medicine|July-September|2023 2 Tannock IF, de Wit R, Berry WR. horti J, Pluzanska A, Chi KN, Ourdard S, Theodore C, James ND, Turesson I, Rosenthal MA, Eisenberger M, and TAX 327 Investigators. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer. The New England Journal of Medicine. 2004;351(15):1502-2. 3 Berthold DR, Pond GR, Soban F, De Wit R, Eisenberger M, Tannock IF. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer: updated survival in the TAX 327 study. Journal of clinical oncology. 2008 Jan 10;26(2):242-5. 4 Bonini SA, Premoli M, Tambaro S, Kumar A, Maccarinelli G, Memo M, Mastinu A. Cannabis sativa: A comprehensive ethnopharmacological review of a medicinal plant with a long history. Journal of ethnopharmacology. 2018 Dec 5;227:300-15. 5 Pellati F, Brighenti V, Sperlea J, Marchetti L, Bertelli D, Benvenuti S. New methods for the comprehensive analysis of bioactive compounds in Cannabis sativa L.(hemp). Molecules. 2018 Oct 14;23(10):2639. 6 Brighenti V, Pellati F, Steinbach M, Maran D, Benvenuti S. Development of a new extraction technique and HPLC method for the analysis of non-psychoactive cannabinoids in fibre-type Cannabis sativa L.(hemp). Journal of Pharmaceutical and Biomedical Analysis. 2017 Sep 5;143:228-36. 7 Thomas A, Baillie GL, Phillips AM, Razdan RK, Ross RA, Pertwee R. Cannabidiol displays unexpectedly high potency as an antagonist of CB 1 and CB 2 receptor agonists in vitro. British journal of pharmacology. 2007 Mar;150(5):613-23. 8 Ligresti A, Moriello AS, Starowicz K, Matias I, Pisanti S, De Petrocellis L, Laezza C, Portella G, Bifulco M, Di Marzo V. Antitumor activity of plant cannabinoids with emphasis on the effect of cannabidiol on human breast carcinoma. Journal of Pharmacology and Experimental Therapeutics. 2006 Sep 1;318(3):1375-87. 9 De Petrocellis L, Ligresti A, Schiano Moriello A, Iappelli M, Verde R, Stott CG, Cristino L, Orlando P, Di Marzo V. Non‐THC cannabinoids inhibit prostate carcinoma growth in vitro and in vivo: pro‐ apoptotic effects and underlying mechanisms. British journal of pharmacology. 2013 Jan;168(1):79-102. 10 Greenhough A, Patsos HA, Williams AC, Paraskeva C. The cannabinoid δ9‐tetrahydrocannabinol inhibits RAS‐MAPK and PI 3 K‐AKT survival signalling and induces BAD‐mediated apoptosis in colorectal cancer cells. International journal of cancer. 2007 Nov 15;121(10):2172-80. 11 Scott KA, Shah S, Dalgleish AG, Liu WM. Enhancing the activity of cannabidiol and other cannabinoids in vitro through modifications to drug combinations and treatment schedules. Anticancer Research. 2013 Oct 1;33(10):4373-80. 12 Nabissi M, Morelli MB, Offidani M, Amantini C, Gentili S, Soriani A, Cardinali C, Leoni P, Santoni G. Cannabinoids synergize with carfilzomib, reducing multiple myeloma cells viability and migration. Oncotarget. 2016 Nov 11;7(47):77543. 13 Li X, Madhukar Kudke A, Joseph Nepveux V F, Xu Y. Networkbased pharmacology study reveals protein targets for medical benefits and harms of cannabinoids in humans. Applied Sciences. 2022 Feb 20;12(4):2205. 14 TÜZÜN B. Investigation of the molecules obtained from marijuana: computational study of spectral, structural and docking. Journal of Physical & Theoretical Chemistry (IAU Iran). 2020;16:59-74. s HOW TO CITE THIS ARTICLE Kaur J, Jabar SIA, Malik AA, Farooqi H, Agarwal S, Panda BP. In-silico and invitro studies of two cannabinoids of Cannabis sativa against prostate cancer. J Ayu Herb Med 2023;9(3):119-124. DOI: 10.31254/jahm.2023.9304 Creative Commons (CC) License- This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY 4.0) license. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. (http://creativecommons.org/licenses/by/4.0/).

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Cannabis sativa, Therapeutic effect, Therapeutic agent, Conflict of interest, Molecular docking, Binding affinity, Secondary metabolite, Drug resistance, MTT assay, Cytotoxic activity, Androgen receptor, Cell viability, Hydroalcoholic extract, Mobile phase, Amino acid, Anticancer properties, Therapeutic dosage, Prostate cancer, IC 50 value, Plant Extract, HPLC system, Leaf extract, Pharmacological effect, Binding energies, Hydrogen bond, Synergistic manner, Brand name, Cytotoxic Agent, Psychoactive Properties, Cell cytotoxicity, Recreational drug, Cannabinoid, Psychoactive Component, Anti-tumor agent, Cancer cell, Extract, Cell multiplication, Prostate cancer cell, CBD, THC.