Microsoft Word - EJBR2023v13i2art114 ISSN 2449-8955 European Journal of Biological Research Review Article European Journal of Biological Research 2023; 13(2): 114-128 DOI: http://dx.doi.org/10.5281/zenodo.8118011 Indigenous plant Cannabis sativa: a comprehensive ethnobotanical and pharmacological review Nilay Vishal Singh, Vinay Kumar Singh * Department of Zoology & Environmental Science, DDU Gorakhpur University, Gorakhpur-273009, UP, India * Corresponding author e-mail: vinaygkpuniv@gmail.com Received: 05 February 2023; Revised submission: 04 April 2023; Accepted: 10 June 2023 https://jbrodka.com/index.php/ejbr Copyright: © The Author(s) 2023. Licensee Joanna Bródka, Poland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/) ABSTRACT: Cannabis sativa (L.) is a plant indigenous to Central Asia and South-East Asia. It is widely used in ethnomedicines as an anti-inflammatory, antioxidant, analgesic, anticonvulsive, antidepressant, anticancer, antitumor, neuroprotective, anti-mutagenic, anti-allergic, and antibiotic. Numerous in vitro and in vivo investigations have already established these attributes of Cannabis. Numerous toxicological studies have demonstrated the dose-dependent toxicity of C. sativa against various pests. The exact identity of the phytoconstituents of C. sativa responsible for the observed biological effects and their mode of action at the molecular level is yet to be ascertained. This review provides a comprehensive update to the ethnomedicinal, phytochemistry, pharmacological activity, and toxicological profile of Cannabis sativa. Keywords: Cannabis sativa; Phytochemistry; Ethnobotany; Pharmacology; Toxicology. 1. INTRODUCTION Cannabis sativa L., (Cannabaceae) also known as Indian hemp, is an annual, herbaceous and dioecious plant with an erect stem reaching up to 5m. It is cultivated in India and China since ancient times [1,2]. Cannabis sativa is possibly among the earliest plants cultivated by man [3,4]. Cannabis has been extensively used by humans as a source of food, fibers, oils, textiles, biofuel, and drugs as well as for religious and recreational purposes over the centuries [5-7]. Almost all parts of the Cannabis sativa are utilized for a variety of use. It contains several phytoactive compounds such as cannabinoids, terpenoids, alkaloids, and flavonoids [8,9]. Cannabinoids are the most active compound, mainly stored in trichomes of female flowers [10,11]. Trans-Δ-9- tetrahydrocannabinol (D9-THC) is the most potent cannabinoid, substantially responsible for psychoactive effects [12]. Natural receptors for THC are found throughout the human body called the ‘endocannabinoid system’ [13]. Cannabis has a rich history of medicinal use dating back to ancient times. Its medicinal uses are described in ancient Ayurvedic text and the first known pharmacopeia “ShenNung Pen Ts’ao Chin” thousands of years ago [14]. Cannabis sativa has been indicated in the treatment of pain, nausea, depression, glaucoma, and neuralgia [15-19]. Cannabis is used for manufacturing fish nets in Artisanal fisheries in the Mediterranean [20] and its fibers are used for manufacturing strings, ropes, fabrics, and papers [21]. Singh and Singh Indigenous plant Cannabis sativa: ethnobotanical and pharmacological review 115 European Journal of Biological Research 2023; 13(2): 114-128 This study aims to provide a detailed account of the taxonomy, phytochemistry, ethnobotanical, pharmacological, and toxicological aspects relevant to C. sativa, so that it may serve as a valuable resource providing future direction for researchers. Cannabis sativa (Indian hemp) (Fig. 1) is a dioecious, sometimes monoecious, annual herb of the family Cannabaceae. The stem is erect reaching up to the height of 5m which also depends on the genetic variety and the environmental conditions [22]. The palmately compound leaves with 5-7 leaflets are alternate or opposite, linear-lanceolate, tapering at both ends and with sharply serrate margins. The male inflorescence is axillary or terminal in a lax panicle and has five pale green hairy tepals (2.5-4 mm), and five anthers. The female flowers are almost sessile and occur in pairs, germinate in the axils, and terminally with one single-ovulate closely adherent perianth. The fruit is an achene, small (2-5 mm long), smooth, light brownish-grey, and a single fruit is produced per flower [23]. Leaves, bracts, and stems of the Cannabis sativa are richly covered by epidermal, glandular trichomes [24,25]. These glandular trichomes contain phytocannabinoids and secondary metabolites [23]. Based on fruit morphology, height, and content of psychoactive molecules, Cannabis is categorized into three species, Cannabis sativa Linnaeus, Cannabis indica Lamarck, and Cannabis ruderalis Janisch [26]. Figure 1.Cannabis sativa (L.).A. Whole plant, B. Male flower, C. Female flower, D. Leaf. 2. ETHNOMEDICINAL USES OF CANNABIS SATIVA Cannabis is perhaps one of the oldest cultivated species for its fiber and food, presumably being utilized for more than 10,000 years [26,27]. It is apparent from archaeological and paleontological records that Europe and Central Asia is the region of the natural origin of Cannabis sativa [28]. The medicinal use of Cannabis was believed to be from China about 5,000 years ago and credit to Emperor Chen Nung, one of the fathers of Chinese medicine [23,29-31]. Cannabis was prescribed for malaria, rheumatism, and fatigue in ancient China [29]. The use of Cannabis as medicine is extensively reported on Assyrian Clay tablets [30] and ancient Egyptian Ebers Papyrus, dated back to 3,000 years ago, which also describes the use of Cannabis for numerous afflictions including infection, analgesia, and vaginal contractions [3,31,32]. During the first and second centuries A.D. Romans cultivated hemp for fibers [33]. Specific mentions Singh and Singh Indigenous plant Cannabis sativa: ethnobotanical and pharmacological review 116 European Journal of Biological Research 2023; 13(2): 114-128 of the remedial use of hemp are found in Arabic literature from the 11th century [34]. Highlighted medicinal properties of hemp include analgesic, intoxicants, narcotic, stomachic, sedative, etc. [35,36]. Cannabis is considered an important medicinal plant in classical Indian Ayurvedic and medicinal texts [37]. Cannabis has been stressed for its Ruchya (taste promoter), Deepan (digestive stimulant), Pachana (digestive), Medhya (memory booster), Madakari (intoxicant), Vyavayi (short-acting), Grahi (withholds secretions), and Rasayana (adaptogen) activities [37]. The foremost written reference to Cannabis sativa in India is found in the Atharvaveda which dates back to around 1500 BCE [38]. Atharvaveda considers it one of the ‘five sacred plants’ [13]. Sushrut Samhita (verses of Sushrut), maybe from the 3rd century BCE, is the direct substantiation of the medicinal use of Cannabis, recommending Cannabis for diarrhoea and phlegm [39]. Hemp is considered a holy plant in Hinduism and used as a form of ‘Ganja’ and ‘Bhang’ to worship lord Shiva, meditation, and communication with spirits [26,40]. Cannabis sativa was used as an organic additive in the clay plaster of the 6th century A.D. caves of Ellora, a World Heritage Site in Maharashtra, India [40]. In the Swat, North Pakistan, leaves of hemp are used in bandages for wound healing, pulverized leaves as an anodyne, sedative tonic and narcotic; juice added with milk and nuts as a cold drink (‘Tandai’) which generates a pleasant excitement and it is also used for Charas preparation [41]. Hemp is a rich source of various narcotics. In India, it is known as Bhang, Charas, Ganja, Marijuana, etc. Despite the number of benefits described over, the production, manufacturing, transport, purchase, and consumption of hemp is illegal and rigorously banned in India. Exceptionally, it can only be used forscientific and medical purposes under the Narcotic Drug and Psychotropic Substance Act in India [42]. 3. CHEMOTAXONOMY OF CANNABIS SATIVA Cannabis sativa is a chemically complex species as it contains diverse groups of phytoconstituents. An aggregate of 565 natural compounds has been identified from Cannabis sativa (Table 2) including 120 cannabinoids, 120 terpenoids (including 61 monoterpenes, 52 sesquiterpenoids, and 5 triterpenoids), 26 flavonoids, and 11 steroids [23, 43-46]. About 120 are identified as phytocannabinoids, because of the shared chemical structure [47]. These phytocannabinoids are lipid-soluble chemical compounds only found in Cannabis exhibiting the typical C21 terpenophenolic skeleton [48]. Table 2. Phytoconstituents and their properties. Chemical class Number of constituents Common constituents Physiological effects Reference D9-THC 23 8α-hydroxy-Δ9–tetrahydrocannabinol, β-fenchyl Δ9 -tetrahydrocannabinolate, α-fenchyl Δ9 -tetrahydrocannabinolate, epi-bornyl Δ9 -tetrahydrocannabinolate, bornyl-Δ9–tetrahydrocannabinolate - partial agonist at both CB1 and CB2 receptors. - psychoactive effects: anxiety, paranoia, perceptual alterations - cognitive deficits - hypolocomotion - hypothermia - catalepsy - analgesia [49] D8-THC 5 Δ8-trans-tetrahydrocannabinol (Δ8- THC), Δ8-trans-tetrahydrocannabinolic acid-A (Δ8- THCA), 10α-hydroxy-Δ8- tetra-hydrocannabinol, 10βhydroxy-Δ8- tetrahydrocannabinol, 10aα-hydroxy-10-oxo-Δ8- tetrahydrocannabinol - a few psychoactive effects on children [50] Singh and Singh Indigenous plant Cannabis sativa: ethnobotanical and pharmacological review 117 European Journal of Biological Research 2023; 13(2): 114-128 Chemical class Number of constituents Common constituents Physiological effects Reference CBG (Cannabigerol) 16 5-acetyl-4-hydroxy-cannabigerol, γ- eudesmyl-cannabigerolate - do not produce psychoactive action mediated by the CB1 receptor. - it is an α-2 adrenergic receptor agonist. - able to inhibit the release of catecholamine with sedation, muscle relaxation, and analgesia effects. - decreases acetylcholine- induced contractions in the human bladder [51-53] CBC (Cannabi- chromene) 9 4-acetoxycannabichromene, 7- hydroxycannabichromane - reduces nitric oxide, IL-10, and interferon-γ levels in peritoneal macrophages activated by LPS - do not show any CB1- mediated psychoactivity. [54,55] CBD (Cannabidiol) 7 cannabidiolic acid (CBDA), cannabidiolmonomethyl ether(CBDM), cannabidiol-C4 (CBD-C4), cannabidivarin (CBDV) - exerts pharmacological effects via specific molecular targets such as glycine receptors, adenosine receptors, serotonin receptors, opioid receptors, nonendocannabinoid G protein-coupled receptors, nicotinic acetylcholine receptors, proliferator- activated receptors. [56] CBND (Cannabinodiol) 2 Cannabinodiol(CBND-C5) and CBND- C3 (cannabinodivarin - unknown biological properties [23,44] CBE (Cannabielsoin) 5 CannabielsoicacidA(CBEAC5-A), cannabielsoin (CBE), cannabielsoic acid B (CBEA-C5 B), cannabielsoic acid B-C3 (CBEA-C3 B) , and C3- cannabeilsoin (CB3-C3) - unknown biological properties [23] CBL (Cannabicyclol) 3 Cannabicyclol (CBL), cannabicyclolic acid (CBLA), and cannabicyclovarin (CBLV) - unknown biological properties [23] CBN (Cannabinol) 11 8-hydroxycannabinolic acid-A and 8- hydroxycannabinol - affinity for CB1 and CB2 receptor is low. [47] CBT (Cannabitriol) 9 -trans-cannabitriol, trans-BT-C5, trans- cannabitriol (-trans-CBT-C5) nd [23] Miscellaneous 30 Dehydrocannabifuran (DCBF-C5), cannabifuran (CBF-C5) nd [23] Cannabinoids 120 Total Non-cannabinoids 445 Cannflavin C, chrysoeriol, 6-prenylapigenin, 4,5-dihydroxy-2,3,6-trimethoxy-9,10-dihydrophenanthrene Total chemicals 565 4. ANTIOXIDANT ACTIVITY An aggregate of 545 compounds has been identified from Cannabis sativa. Generally, phenolic compounds (flavonoids) are responsible for antioxidant activity [46]. Drinic et al., analyzed antioxidant the activity of extracts of C. sativa using DPPH (2,2’-diphenyl-1-picrylhydrazyl) assay,and antioxidant activity was expressed through EC50 values ranging from 0.1331 mg/ml to 0.4353 mg/ml and 0.2055 mg/ml to 0.7563 mg/ml for aerial parts of young and mature hemp, respectively [57]. Wang et al., and Girgih et al. reported that proteins isolated from Cannabis could be effectively hydrolyzed by Neutrase and these hydrolysates exhibit antioxidant activity determined by DPPH assay and Fe2+ chelating capability [58,59]. Hydrolysates showed DPPH radical Singh and Singh Indigenous plant Cannabis sativa: ethnobotanical and pharmacological review 118 European Journal of Biological Research 2023; 13(2): 114-128 scavenging activity (IC50) ranging from 2.3 mg/ml to 3.5 mg/ml for different time intervals. Antioxidant assays of hemp seeds including FRAP, DPPH, and Chelating power assays suggest significant antioxidant capacity [60-62]. C. sativa seeds contain mainly storage proteins (albumin 25-37%, legumin 67-75%) and show antioxidant activities against human keratinocytes [63]. The antioxidant activity of hemp determined by reducing power method using solvent extracts of different polarity at different extraction times was found to range between 0.2025 to 0.866% for acetone and methanol extracts, respectively [64]. Lignanamides (cannabisin M, cannabisin N, cannabsin O, and 3,3’-dimethyl-heliotropamide) isolated from Hemp seeds show antioxidant and acetylcholinesterase inhibition activities [65]. Hemp seed extracts increase gene expression of antioxidant enzymes (SOD, glutathione peroxidase, and catalase) in a concentration-dependent manner in H2O2- challenged HepG2 cells [66]. Hemp seed oil at different concentrations improves the oxidative state of Drosophila melanogaster under non-stressed as well as H2O2-induced stress conditions [67]. C. sativa essential oils exhibit antioxidant activity with IC50 values of 1.6 mg/ml for DPPH assay (free radical scavenging activity), 1.8 mg/ml for β-carotene/linoleic acid assay, and 0.9 mg/ml for reducing power assay [68]. Ahmed et al. reported that different organic solvent extracts of C. sativa leaves showed DPPH inhibition activities ranging from 34.2% to 55.57% [69]. Stem, leaves, and inflorescence of male and female hemp plants exhibit high ABTS (2,2’-azinobis (3- ethylbenzothiazoline-6-sulfonic acid) radical cation scavenging activity, DPPH assay, TPC (Total Phenolic Content) assay and metal chelating activity [70]. C. sativa var. future inflorescence extract was used to determine the antioxidant activity in vitro and on an ex vivo model of human erythrocytes [71-73]. Cannabis fibrant extracts obtained from the inflorescence of a non-psychotropic variety of hemp (C. sativa var. fibrant) show a strong and concentration-dependent antioxidant activity [74]. Singh et al. screened the methanolic extract of hemp for antioxidant activity with DPPH assay value of 45.8% [75]. Lignanamides, cannabisins, 3,3’-demethylgrossamide, and N-trans-caffeoyltyramine extracted from Cannabis were evaluated on the mammalian arginase assay, which showed that N-trans-caffeoyltyramine exhibited higher antioxidant activity with IC50 value of 20.9 µM [76]. Green synthesized silver nanoparticles of hemp leaves were used for antioxidant activity evaluation using ABTS and DPPH assays, and results ranges from 73.109% to 90.33% inhibition and 1.506% to 6.670 % inhibition for ABTS and DPPH assays, respectively [77]. Green synthesized gold nanoparticles of hemp leaves showed antioxidant activity. IC50 values of DPPH assay for leaf crude extract and gold nanoparticles were 361 µg/ml and 196 µg/ml, respectively [78]. The DPPH radical inhibition percentage shown by green synthesized ZnO nanoparticles from hemp leaves was 88% at 1500 µg/ml [79]. Whilst these findings reveal the good antioxidant potential of C. sativa, further in vivo studies are warranted, particularly focusing on how these plant constituents may interfere with the antioxidant enzymes. 5. ANTI-CANCER ACTIVITY Over the past years, several studies on cell cultures and animal models have demonstrated that phytocannabinoids possess potential anti-cancer properties, including the inhibition of invasion and metastasis, angiogenesis,and promote apoptosis in cancer cells [14,80-84]. Cannabinoids and their derivatives exert palliative effects in cancer patients by preventing nausea, vomiting, pain and appetite stimulation, and attenuation of wasting [85]. The discovery of the anti-cancer effect of cannabinoids can be dated back to Munson et al., who demonstrated that the administration of Δ9-tetrahydrocannabinol (Δ9-THC), Δ8-tetrahydrocannabinol (Δ8-THC) Singh and Singh Indigenous plant Cannabis sativa: ethnobotanical and pharmacological review 119 European Journal of Biological Research 2023; 13(2): 114-128 and cannabinol inhibited the growth of Lewis lung adenocarcinoma cells in vitro and in vivo after oral administration in mice in a dose-dependent manner [86,87]. Besides cannabinoids, terpenes and flavonoids have also been shown to exert anti-tumorigenic actions. Δ9-THC is a major psychoactive compound present in Hemp which mediates its effect by binding and activating CB1 receptors in the central nervous system [88]. THC at a concentration of 14 µM inhibited overall cell growth in the MCF-7 cell line [89]. In breast cancer cells, THC inhibited the cell cycle progression at G2/M phase by down-regulating Cdc2, and induced apoptosis via CB2 receptor [90]. McAllister et al., reported that THC at a concentration of 1.2 µM/L was able to reduce the proliferation and invasion of breast cancer cells via reducing the expression of Id-1 transcriptional factor [91]. Takeda et al., found that Δ9-THC inhibited estrodiol-induced cell proliferation by inhibiting estrogen receptor α (ER α) activation [92]. Sharma and Sharma reported that green synthesized Cr2O3 nanoparticles from C. sativa leaf extracts possessed potent anti-cancer activity against HepG2 cancer cell line [93]. Green synthesized Ag and ZnO nanoparticles from callus extract of C. sativa showed promising cytotoxic potential against HepG2 cell lines [94]. Chang et al. reported that the gold- nanoparticles synthesized using aqueous extracts of C. sativa leaf can be used for the therapy of acute lymphoblastic leukemia and T-cell leukemia [78]. Go et al., found that Cannabidiol (CBD), which is one of the identified cannabinoid in the C. sativa, has anti- cancer potential as a cytotoxic drug for Head and Neck Squamous Cell Carcinoma (HNSCC) and combinational treatment of CBD enhanced the efficacy of chemotherapeutic drugs [95]. Results obtained by treating human prostate cancer cell lines LNCaP, DU145, and PC3 with Cannabidiol (CBD) clearly indicate that CBD is a potent inhibitor of cancer cell growth and induces apoptosis in a dose-dependent manner [96]. Cannabichromene (CBC) from C. sativa extracts, in combination with CBD and THC, was found to induce apoptosis, cell-cycle arrest, inhibited cell migration, and affected F-actin integrity [97]. Methanolic extracts of C. sativa exhibit potent anti-cancer activity with an IC50 value of 8.63 µg/ml against the colon cancer cell line, whereas, CBC and THC have IC50 values of 6.06 and 14.33 µg/ml, respectively [98]. In contrast, THC was shown to stimulate cancer cell proliferation in breast cancer and MCF-7 cell by increasing Human Epidermal Growth Factor 2 (HER2) expression [92]. Similarly, Mckallip et al., reported that Δ9-THC exposure led to increased 4T1 mammary carcinoma growth and metastasis in mice [99]. 6. ANTI-MICROBIAL ACTIVITY An in vitro disk diffusion assay was carried out to evaluate the antimicrobial activity of the essential oils from three varieties of Cannabis sativa (Carmagnola, Fibranova and Futura) against various Gram-positive (Clostridium bifermentas, C. butyricum, C. sporogenes, C. tyrobutyricum, Enterococcus hirae, E. faecium, Streptococcus salivarius) and Gram-negative (Pectobacterium carotovorum subsp. carotovorum, Pseudomonas corrugata, P. fluorescens, P. savastonoi pv. phaseolicola, P. syringae pv. atrofaciens, Pseudomonas viridiflava, P. campestris pv. pruni) bacteria. All extracts dose-dependently inhibited these bacteria [100]. Another study showed that the aqueous and acetone extracts of the plant were active against bacteria Pseudomonas aeruginosa, Vibro cholerae, and fungi Cryptococcus neoformans, Candida albicans. The minimum inhibitory concentrations (MIC) of these extracts against the microorganisms were in the range of 5 µg/ml and 10 µg/ml [101]. Novak et al. evaluated the antibacterial activity of the essential oils of five different cultivars of C. sativa (SwissMix, Felina 34, Fedrina 74, Kompolti, Secuemi) against a large panel of Gram-negative and Gram- positive pathogens with only modest activity against Acinetobacter calcoaceticus and Brevibacterium linens [102]. The heat-treated retted, semi-retted, and non-retted hemp hurd powder (at 160°C for 2 h) showed efficient antibacterial activity up to 90% reduction in CFU against E. coli [103]. Another study showed that minimum Singh and Singh Indigenous plant Cannabis sativa: ethnobotanical and pharmacological review 120 European Journal of Biological Research 2023; 13(2): 114-128 inhibitory concentrations (MIC) of cannabidiol (CBD) from C. sativa against Salmonella newington and S. typhimurium was 0.0125 μg/ml and 0.125 μg/ml, respectively [104]. The ethanolic and petroleum ether extracts of the leaves of Cannabis sativa exhibited antimicrobial activity against Gram-positive (Baccilus subtilus, B. pumilus, Staphlococcus aureus, Micrococcus flavus), Gram-negative bacteria (Proteus vulgaris, Bordetella bronchioseptica) and fungi (Candida albicans, Aspergillus niger) [105]. Satyal and Setzer screened the essential oil from Nepalese Cannabis sativa for antimicrobial activity with the Minimum Inhibitory Concentration (MIC) of 625 µg/mL for Bacillus cereus, 2500 µg/mL for Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, 625 µg/mL for Aspergillus niger and 1250 µg/mL for Candida albicans [106]. Another testing of the Cannabis fibrant Hexane Extract 1 (CFHE1) and C. fibrant Hexane Extract 2 (CFHE2) on Staphylococcus aureus and methicillin- resistant clinical strains led to the modest inhibitory effect with Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of 4.88 μg/ml for CFHE1, and 4.88 and 19.53 μg/ml, respectively, for CFHE2 against Staphylococcus aureus and MIC between 1.22 and 9.77 μg/ml and MBC between 4.88 and 78.13 μg/ml for methicillin-resistant S. aureus (MRSA) using vancomycin as a standard [74]. Singh et al. reported the antibacterial activity of the green synthesized Ag-nano particles from C. sativa against Pseudomonas aeruginosa and Escherichia coli with MBC values of 12.5 and 25 µg/mL and MIC values of 6.25 and 5 µg/mL against P. aeruginosa and E. coli, respectively [107]. Ag nanoparticles prepared from leaf extracts of C. sativa exhibited the highest antibacterial potential against Escherichia coli and Micrococcus luteus [108]. Additional testing of bactericidal activity of Ag-NPs and ZnO-NPs synthesized from callus extract of C. sativa showed that Ag-NPs exhibited the highest activity against Staphylococcus epidermidis and Pseudomonas aeruginosa with 14 mm and 12 mm zone of inhibition, respectively and ZnO-NPs showed highest activity against Klebsiella pneumoniae, Pseudomonas aeruginosa and Bacillus subtilis with an inhibition zone of 20 mm, 18 mm, and 15 mm, respectively [94]. Overall, C. sativa extracts have demonstrated in vitro activity against selected microorganisms, which should be further investigated particularly by employing in vivo models of infection and the molecular mechanisms underlying the antimicrobial activity of C. sativa extracts/constituents should also be elucidated. 7. PESTICIDAL ACTIVITY There are some evidences that Cannabis sativa possesses pesticidal activity. The toxicity of essential oils was evaluated against larvae of Hyalomma dromedarii and Dermanyssus gallinae which showed LC50 values of 73 µg/mL and 47.1 µg/mL, respectively [109]. Petroleum ether, Carbon tetrachloride, and methanol extracts of C. sativa have been evaluated against the Culex quinquefasciatus larvae. The LC50 values of Petroleum ether, Carbon tetrachloride, and methanol extracts were 294.42 ppm, 88.51 ppm, and 160.78 ppm, respectively, after 24h of exposure [110]. Another observation showed that the LC50 value (24h exposure) of essential oil of C. sativa against Culex quinquefasciatus was 127.3 µg/ml [111]. Rossi et al. evaluated the toxicity of C. sativa essential oils and observed that LC50 values were ranging from 73.50 to 78.80 ppm for larvae and 20.13 to 67.19 ppm for pupae of Anopheles stephensi and A. gambiae, respectively [112]. Benelliet al. evaluated the toxicity of essential oils from the inflorescence of industrial hemp (C. sativa) cv Felina 32 and found that it was highly toxic to aphid Myzus persicae adult ((LD50 = 3.5 ml/L) and Musca domestica flies (LD50 = 43.3 μg/adult) and moderately toxic towards Spodoptera littoralis larvae (LD50 = 152.3 μg larva−1) and C. quinquefasciatus 3rd instar larva (LC50 = 252.5 μL/L) [113]. Additionally, Bedini et al. evaluated the toxicity of essential oils of C. sativa against larvae of Aedes albopictus, adults of Physa marmorata, and nymphs of Cleon dipterum with LC50 values of 301.56, 282.17 and 35.37 μL/L [114]. C. sativa essential oil was found Singh and Singh Indigenous plant Cannabis sativa: ethnobotanical and pharmacological review 121 European Journal of Biological Research 2023; 13(2): 114-128 to be toxic against Reticulitermes virginicus with LC50 354µg/mL [106]. It is evident from these studies that Cannabis sativa can be potentially used as biopesticides. Further, detailed investigations should be carried out in the future to discover new plant-based chemical entities that could replace the harmful commercial pesticides. 8. ACUTE AND CHRONIC TOXICITY OF CANNABIS ON HUMANS Acute Cannabis use negatively impacts memory and learning abilities. For instance, acute cannabis intoxication has frequently been reported to result in noticeable changes in subjective mental state with detrimental effects on neuropsychological functioning, including learning performance and resulting in decreased attention and working memory [115-118]. Pope found that extensive Cannabis usage was linked to lower attentional/executive system performance, as seen by lower mental flexibility, higher perseverance, and lower learning [119]. Investigations of acute effects among non-severe heavy users, higher concentrations of the psychoactive compound tetrahydrocannabinol (THC) have been linked to deficits in planning and control impulse activities, with effects lasting for up to four weeks after drug use. [120] Compared to non-users Cannabis users demonstrated deficits on, visual recognition, verbal skills, delayed visual recall, and short- and long-interval prospective memory tasks [121]. In regular marijuana users, marijuana's immediate effects may have an impact on a person's cognitive-motor abilities and the brain mechanisms that control driving and coordinated movement in regular marijuana users [122]. Addiction is one of the most detrimental effects of persistent Cannabis use. The emotional reactions to cannabinoids are not always positive and delightful. Instead, CB1 receptor activation may also result in anxiety and panic [123]. Diagnoses of depression, along with anhedonia, agoraphobia, and suicidal ideation, were found to be significantly linked to an increased likelihood of cannabis use [124-126]. A subgroup of cannabis users may be more susceptible to developing chronic psychoses like schizophrenia due to genetic vulnerability [127]. 9. CONCLUSION Cannabis is an ethnobotanically rich and phytochemical significant therapeutic plant. Various ancient literature and multiple in vitro and in vivo studies on Cannabis sativa have demonstrated its remarkable medicinal potential. C. sativa is enriched with significant antimicrobial and antioxidant activity due to its active phytoconstituents like cannabinoids, phenolic compounds, and tannins. Investigations have revealed the toxicity of the phytoconstituents of C. sativa. 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