Hemp Growth Factors and Extraction Methods Effect on Antimicrobial Activity of Hemp Seed Oil: A Systematic Review

: The bioactive Hemp Seed Oil (HSO) is becoming very popular in the medical and research fields due to its antimicrobial properties against several diseases caused by bacteria and fungi. How ‐ ever, the effect of hemp ‐ growing factors and extraction methods on the bioactivity of HSO does not receive adequate research attention. Therefore, this review aims to investigate the effect of growth factors and extraction methods on the antimicrobial activity of HSO. Articles were retrieved from Google Scholar and the Scopus database and screened against inclusion and exclusion criteria. The study revealed that HSO prefers warm climates and favorable humidity ranging from 20 to 39 °C and 79–100% per year, respectively, and rainfall of 324 mm daily. The multivariate linear regression shown excellent prediction (R 2 = 0.94) with climates upon Zone of Growth Inhibition (ZGI) of Gram ‐ positive bacteria. Temperature is the strongest predictor ( p < 0.01) followed by humidity and rainfall ( p < 0.05). Furthermore, well ‐ drained loam soil rich in organic matter seems to stimulate the antimi ‐ crobial activity of HSO. The major constituents that influence HSO’s antimicrobial ability to Staph ‐ ylococcus aureus were cannabidiol (CBD), β‐ caryophyllene, and limonene. The extraction methods showed less influence on the HSO bioactivity. HSO did not show significant antioxidant activity, but Hemp Seed Hull (HSH), Hemp Seed Flour (HSF), and Hydrolyzed Hemp Seed Protein (HPH), expressed promising DPPH scavenging ability.


Introduction
The hemp plant is considered one of the world's ancient cultivated plants that was developed from wild Cannabis plants originated from Central Asia and has long been widely used for textiles and as a nutritional source of food, cosmetics, and oriental medicine [1,2]. Hemp is also known as a short-day and photoperiod-sensitive crop that exhibits several nutraceutical properties to produce cannabinoids, fatty acids, and other nutritional products for biomedical uses [3]. It is also an active ingredient in commercial insect repellents and biopesticides assigned to its antioxidant and antimicrobial properties [4,5]. Furthermore, hemp is a dioecious annual multipurpose crop grown for its seed, oil, food, and medicinal properties [6]. Hemp seed originates from the mature female Cannabis sativa L. plant constituting 25-35% lipid, 20-25% protein, and 30% carbohydrates, as reported by Deferne and Pate [7]. In addition, hemp seeds are used for the preparation of animal feed, oil, flour, and protein powder. In recent years, Hemp Seed Oil (HSO) has received public attention in its usage due to its valuable addition to human and animal diets. The hemp plant's seed oil contains anti-inflammatory properties due of its ideal ratio and high quantity of omega-6 (Linoleic Acid (LA)) to omega-3 (Alpha-Linolenic Acid (ALA)) Polyunsaturated Fatty Acids (PUFAs), 3:1, along with other PUFAs such as Gamma-Linoleic Acid (GLA) [8]. Furthermore, HSO possesses promising antimicrobial and antioxidant properties because of the plant's content in tocopherols (100-150 mg/100 g oil), terpenes, phenolic compounds, cannabinoids, vitamins, and minerals [9,10].
Out of all the plant's chemical compounds, cannabinoids represent the most studied group mainly because of their numerous pharmaceutical effects in humans [11]. More than 100 different chemical compounds called cannabinoids can be extracted from hemp as reported by García-Tejero et al. [12]. Many studies suggest that cannabidiol (CBD) has potential antimicrobial effects, especially against drug-resistant strains of Staphylococcus aureus [13][14][15]. Although HSO does not traditionally contain cannabinoids as it is found on the plant's leaves and flowers, an alternative hemp oil called Hemp Essential Oil (HEO) could be used to utilize a variety of the plant's beneficial components. HEO is defined as a complex mixture of several volatile compounds, mainly monoterpenes, sesquiterpenes, and other terpenoid-like substances [16]. It is extracted from the plant's leaves, flowers, seeds, seed bracts, inflorescences, and thinner stems [16]. Zheljazkov et al. [16] found that HEO, containing the highest CBD, expressed the highest antimicrobial inhibition against Gram-positive bacteria. The two most abundant cannabinoids in the hemp plant are tetrahydrocannabinol (THC) and CBD, which are most popular for their therapeutic and psychotropic effects. The hemp plant contains 12-18% of CBD on average and 0.3% of THC, while the seed only contains a moderate amount of CBD and THC [17]. Fathordoobady et al. [18] concluded that the non-psychoactive cannabinoids such as CBD are present mainly in seed oil, while most THC accumulates in plant leaves. It is possible that the CDB presence in HSO could be caused by the hemp plant's contact with the resin secreted by the epidermal glands situated on flowers and leaves [19].
The chemical constituents of HSOs are tocopherols, terpenes, phenolic compounds, Vitamin B complex, sodium, calcium, iron, sulfur, potassium, phosphorus, zinc, and copper [2,20]. Due to the composition of these natural antioxidants in HSO, it was believed to have promising antioxidant effects. For example, the tocopherol acts as an antioxidant to prevent the oxidation of unsaturated fatty acids, as reported by Kriese et al. [21]. Unfortunately, at the end of this study, it was concluded that HSO did not perform as a good antioxidant; however, a more broad study would need to be completed in order to detect what influences the antioxidant activity in HSO.
The quality and the quantity of the bioactive components of the HSO depend on field conditions in which the hemp is being grown, the crop variety, and the extraction process used to obtain it [22]. The hemp's growth, yield, nutritional, and bioactive constituents depend on the interaction between the hemp variety, environment, and management [23]; here, plant density, soil fertility, and climatic conditions (rainfall, temperature, and humidity) are the main factors affecting hemp chemical compounds [24][25][26]. The interaction of these factors affects plant development, which will eventually result in low bioactive constituents of HSO. Togliatti's [27] study shows that growing conditions such as temperature, precipitation, and radiation affect hemp productivity. Poor soil and climatic conditions hinder the growth of hemp by restraining the root development and nutrients constituents, especially when the soil is acidic and compacted [1]. On the contrary, many research findings indicated that hemp plants could survive harsh climatic conditions and poor soil environments. Therefore, some critical results regarding how the climatic conditions, soil environment, and bioactive compounds' extraction methods contribute to HSO's antimicrobial and antioxidant activity have not been explicit. Hence, this paper aims to elucidate these key gaps identified in the literature to provide an unambiguous end to these issues. Furthermore, this will give hemp cultivators a better understanding of the ideal temperatures, humidity, and water content that the plant needs for optimal nutritional benefits.
In this paper, we systematically review and organize the bioactivity of HSO literature toward the following objectives:


To elucidate the effects of hemp's growth factors (temperature, rainfall, and humidity soil types) on the antimicrobial activity of hemp oils using existing evidence from peer-reviewed articles.  To find out the various existing extraction methods on the antimicrobial activity of hemp oils.  To provide the updated existing literature on the hemp-derived products' antimicrobial and antioxidant effects.

Search Procedures
HSO has been documented to contain bioactive properties. Many factors influence the components that express these properties, including extracting the oil, major constituents, and climate. The systematic review was gathered through a literature search from online databases. Relevant articles were searched on Google Scholar and the Scopus database to identify how climatic conditions and extraction methods affect Hemp Seed Oil's bioactivity. Boolean operators "AND" and "OR" were used to broaden the search. Some of the keywords used for searching were temperature AND bioactivity of HSO, antimicrobial AND antioxidant effects of HSO OR other hemp oils, etc. The hemp location, temperature, rainfall, and humidity were researched [28]. The soil was identified through the Scopus database and Google Scholar online. Since the bioactivity of the HSO literature is so diverse, we supplemented our search with citations in recent studies (2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014)(2015)(2016)(2017)(2018)(2019)(2020). To further ensure that we had assembled a comprehensive list of studies, we asked researchers with the relevant knowledge on the topic to review and suggest keywords.
The search focused on scientific research articles using the following protocol: i. Publication years were between 2004 and 2020; ii. The keywords ''hemp oil" AND ''antimicrobial activity"; "hemp oil and climatic indicators"; hemp extraction method AND antimicrobial activity had to appear in the title and abstract; iii. They had to be scientific indexed papers only.
The results were screened against inclusion criteria i.e., articles that are not relevant to the studies. Full text of papers for all the articles that fitted into the inclusion criteria was retrieved.

Screening
Strict criteria were used to determine the relevant articles for inclusion. For example, articles were being excluded if published in languages other than English or for which only an abstract was available, and then each remaining search result was grouped as one of the three publication types (criteria), which were adopted and modified by Gurwick et al. (2013) [26]: i.
"Primary articles" Research papers appeared in the peer-reviewed literature and reported original data or results based on observations and experiments. ii. "Methods" papers evaluated or described an investigative technique for the extraction of chemical compounds of hemp oil. iii. "Review" papers summarized the understanding of bioactivity of hemp oil and antimicrobial activity [26].
Throughout the screening process, the number of publications excluded in each stage and their reasons for exclusion were noted based on the guidelines outlined in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statement [29] in Figure 1.

Data Extraction and Reporting
A standard, purpose-designed form was designed to extract data (location, method of HSO extraction, antimicrobial activity, soil type and hemp product types) from the paper. Hemp antimicrobial activity is affected by the plant variety and growth stage of harvesting. However, none of the articles reported on the variety of hemp plant used, the type of fertilizer applied, and the period of harvesting of the hemp.
Nineteen papers were identified and reviewed on Google Scholar and Scopus for their antioxidant and antimicrobial properties. The articles retrieved range from 2004 to 2020. Eleven papers were compared to identify hemp oil's antimicrobial activity. Four papers were based on HSO, three were based on HEO, two were based on cannabinoids in oil, and two were based on Hemp Leaf Extract (HLE).
The oils were tested against the seven Gram-positive bacteria: Based on these eight locations, temperature, rainfall, humidity, and soil type were also analyzed. Temperature, rainfall, and humidity were investigated with multivariable linear regression. The average yearly/annual climate data were collected from Weath-erSpark n.d. [28]. The average minimum and maximum temperature (°C) were taken from the months February, March, July, and October. Average rainfall (mm) was taken from the months February, March, July, October, and November using the formular: The average humidity (%) of the start and end of each month was taken from the months February, March, July, October, and November. The relation between average temperature, rainfall, and humidity with the ZGI of Gram-positive bacteria was calculated via Excel (Figure 2d). Major constituents and extraction methods were also analyzed in regard to the antimicrobial potential of hemp products. Ten out of eleven papers included the extraction method used to extract the oil from the plant. A table was put together to compare the antimicrobial potential and the extraction method used. Four papers out of eleven gave the plant's major constituents [16,[30][31][32]. The antioxidant activity of HSO was observed through radical scavenging activity against 2,2-diphenylpicrylhydrazyl (DPPH). The radical scavenging activities of hemp oils and hemp products were investigated and were with each other, other oils, and their controls.  Figure 2d shows multivariate linear regression to find the relationship between the ZGI with temperature, rainfall, and humidity. The linear regression showed the climate on prediction of ZGI (R2 = 0.94). The prediction equation: Inhibition zone = 0.0723 + 0.8089 × Temperature − 0.1982 × Rainfall + 0.3334 × Humidity. Temperature is the strongest predictor (p < 0.01) followed by humidity and rainfall (p < 0.05).

Hemp-Derived Products' Major Constituents Influence on Antimicrobial Activity
For HSO to obtain high bioactive properties, it should be high in cannabinoids, monoterpenes, terpenes, tocopherols, and phenolic compounds. Eleven papers out of the total nineteen papers reviewed tested for hemp-derived products' antimicrobial effects and the summary of the antimicrobial effects of hemp-derived products is presented in Table 1.
Four out of these eleven papers gave the plant oils' major constituents, which were compared and observed to show which compounds performed the best in regard to antimicrobial activity. HSO and HEO were observed. The major components found in HSO were myrcene, b-sitosterol, γ-tocopherol, CBD, β-caryophyllene, and a trace amount of αtocopherol and methyl salicylate [32]. Other major constituents found in HEO were αcaryophyllene, caryophyllene oxide, limonene, β-(E)-ocimene, and a-pinene. More constituents are likely to be found in HEO rather than HSO because HEO contains various parts of the plant to produce the oil, such as the leaves, flowers, seeds, seed bracts, inflorescences, and thinner stems.
CBD is known to be a plant-derived cannabinoid (phytocannabinoid) that occurs uniquely in hemp plants [33]. In this current study, CBD, in particular, had a commonality within three of the papers that ranged between 0 and 52% in content. CBD, along with other cannabinoids, has been reported to be antibacterial, specifically against Staphylococcus aureus [13,15]. HEO from the 'wild Buro' and 'wild Saykaj' plants showed the most antimicrobial activity against all Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, Streptococcus pneumoniae) and Gram-negative bacteria (Salmonella enterica, Pseudomonas aeruginosa) compared to all other HEO, which contained less (Table 1). Similar studies conducted by Russo and Reggiani, 2013, concluded that the bioactive compounds in hemp plants are influenced by the hemp variety. However, none of the articles reported on the hemp age, variety, growth conditions (nutrition, humidity, and light levels), harvest time, and storage conditions, making it difficult to deduce a proper conclusion [34].
Specifically, the CBD antimicrobial activity against Gram-negative bacteria depends on lipopolysaccharides (LPS) and the type or the species of the Gram-negative bacteria [16,Zhel CBD]. In addition, different types of LPS can be found in different genera of Gram-negative bacteria, contributing to 80% of the outer membrane of the Gram-negative bacteria. Therefore, the higher the lipopolysaccharides of the Gram-negative bacteria, the lower the possibility of CBD expressing its antimicrobial activities against the Gram-negative bacteria diseases or the higher the bacterial virulence [35]. HSO only expressed 10 mg/kg of CBD and did not exhibit much activity against Gram-negative bacteria due to the most abundant antigen (lipopolysaccharides) in the outer membrane of the most Gram-negative bacteria causing resistance to the CBD antimicrobial activity to inhibit the cell wall biosynthesis of the bacteria as well as the low amount of CDB contained in the HSO [36]. However, the amount of the CDB extracted from the HSO depends on the hemp age, variety, growth conditions (nutrition, humidity, and light levels), harvest time, and storage conditions, as reported by Sáez-Pérez et al. [22].
Moreover, oil derived from the hemp's seed did not exhibit any antifungal activity [9 Ali]. Whereas, oil extracted from the whole hemp plant did present moderate activity against fungi because of the high CBD, cannabigerol (CBG), and cannabichromene (CBC) present in the whole hemp plant oil [9]. Monoterpenes such as myrcene, α-pinene, and limonene against Escherichia coli, S. Enterica, and Staphylococcus aureus have shown some antibacterial properties, which are most likely assigned to their diastereomeric structure, particularly limonene against Gram-positive bacteria [37,38]. Limonene is most concentrated in HEO grown in India, ranging from 4.1 to 15.8%. The highest concentration of compounds (limonene, α-pinene, terpinolene, myrcene, and β-(E)-ocimene) was observed in October, suggesting that the best harvest time to obtain antimicrobial properties is in October. Myrcene and α-pinene are most concentrated in the HEO from Backi Petrovac, Serbia with 9.79-16.33% and 10.7-20.7% concentration [30]. Correspondingly, β-myrcene has shown antimicrobial potential in other studies [39,40]. All HEOs exhibited moderate to low activity against Gram-positive and Gram-negative bacteria. Sesquiterpenes found in hemp oils such as β-caryophyllene, caryophyllene oxide, αhumulene, and δ-cadinene have also been reported to halt microbial growth [44,45]. (β)-Caryophyllene has shown antibacterial activity against Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Aspergillus niger in the past [46], caryophyllene oxide against Staphylococcus aureus and Enterococcus faecalis [47], α-humulene against Bacillus cereus, Staphylococcus aureus, and Aspergillus niger [45], and δ-cadinene against Bacillus subtilis. Not only does β-caryophyllene show exceptional inhibitory results against bacteria and fungi, it also serves as a good antioxidant and anticarcinogenic terpene [46]. Zheljazkov et al. [16] exhibited the most βcaryophyllene out of 22.4-55%, which would correspond with its highest antimicrobial effect compared to the other three papers [16]. The result is similar to the findings of [48], as they concluded that the presence of sesquiterpenes or the presence of CBD could have an antimicrobial effect on the oil extracted from Cannabis Sativa.
Based on the Mikulcová et al. [4] study, it was concluded that the higher content of ALA could explain the higher antibacterial activity in unrefined oils compared to refined oils [4]. In addition, it was also proposed that because tocopherols and tetrahydrocannabinol are removed during the refining process, they could indicate that they pertain to antimicrobial potentially in unrefined oils. It was also noted that Gram-positive bacteria were more sensitive to unrefined oils compared to Gram-negative strains because of Gram-positive bacteria's lack of a lipopolysaccharide outer layer. Gram-negative Micrococcus luteus and Staphylococcus aureus were proved the most sensitive to the oils, while Escherichia coli proved most resistant.

Climatic Indicators (Temperature, Rainfall, and Humidity) on Antimicrobial Activity of Hemp Seed Oil
Figure 2a-c show that India, Sudan, Nigeria, and Pakistan express the highest temperatures, rainfall, and humidity levels. In relationship, the highest antimicrobial growth occurred in hemp oils sourced from Sudan, Nigeria, and Pakistan [9,42,43]. Therefore, the rationale underpinning this objective is that if hemp plants experience optimal growing conditions of mild climates, humid atmosphere, and a rainfall of at least 635-762 mm per year, the plants will obtain high quantities of antimicrobial compounds. HSO cultivated in Niala, South Darfur, and Sudan possessed the highest activity against Gram-positive Staphylococcus aureus with 28 mm of ZGI. HSO did not show any activity against fungi, which may be due to the low bioactive component in the hemp seed, such as CBD CBG and CBC, causing low inhibition of ergosterol synthesis in fungi [9]. However, hemp oil derived from the whole hemp plant exhibited moderate antifungal activity against Aspergillus niger and Candida albicans due to their high CBD, CBG, and CBC, as reported by Ali et al. [9]. Its temperature was high, ranging from 15 to 39 °C (Figure 2a). The rainfall observed was higher in July (98 mm) (Figure 2b). Humidity increased from 0% in February to 79% in July, and it was 0% again in October (Figure 2c).
HSO from the hemp plant grown in Mansehra, Pakistan showed the most significant inhibitory effects against Gram-negative bacteria, particularly against Escherichia coli (22.2 mm) and Pseudomonas aeruginosa (25.3 mm). This may be due to the conducive growth condition (rainfall and fertile soil) of the hemp in the region, contributing to the high amount of CDB, causing high inhibition of cell wall biosynthesis of the Gram-negative bacteria [9].
Pakistan's temperatures ranged from 0 °C in February to 31 °C in May and July (Figure 2a). The rainfall obtained by Pakistan was similar to the rainfall observed in Sudan, ranging from 16 to 131 mm a day (Figure 2b). Pakistan was the only location where the rainfall was high in February, decreased in May, and increased again in July (Figure 2b) [42]. The second most increased activity against Gram-negative bacteria was seen in HLE, in Nigeria, which may be assigned to the favourable growth conditions in the location needed for the hemp growth development. Mediavilla et al. [23] concluded that strong antimicrobial activity depends on tissue type, age, variety, growth conditions (nutrition, humidity, and light levels), harvest time, and storage conditions. However, none of the articles reported on the hemp age, variety, growth conditions (nutrition, humidity, and light levels), harvest time, and storage conditions, making it difficult to deduce a proper conclusion.
In Figure 2a-c, Nigeria possessed the highest climates, humidity, and rainfall compared to the other locations. The lowest Gram-negative bacteria activity was found in the Czech Republic, which was followed by India with only activity against Salmonella typhimurium and no activity in Escherichia coli, K. Pneumoniae, or Pseudomonas aeruginosa. Prague's temperatures ranged from −3 °C to 25 °C, rainfall ranged 15 mm to 64 mm a day, and humidity was only as high as 2%. The results obtained could be assigned to the possibility that the hemp plant was cultivated in the greenhouse, where temperature, humidity, and water content were highly ensured.
Nigeria obtained the highest temperature throughout the year, ranging from 22 °C to 31 °C, with the lowest and highest end of the range recorded by February and October. The temperature recorded in October was indicated as the highest compared to the other countries ( Figure 2a). In addition to the high humidity of 93% to 100%, high rainfall of 40-324 mm daily, high soil fertility, and high content of CBD, the hemp leaf oil possessed the most increased antifungal activity up to 40 mm of growth inhibition against Candida albicans. The hemp plant that shows the highest activity against Gram-negative bacteria in Pakistan was similar to the hemp plant grown in Nigeria [42,43]. The hemp plant was raised in an environment with high temperatures and humidity of 27 °C and 80%, respectively (Figure 2a,c). Both of these oils were extracts from leaves and showed similar results. However, the oil from Pakistan was not tested against fungi; therefore, it is unknown how much the climatic conditions affect the hemp plant's antifungal properties [42]. The leaf contains a large percentage of the plant's cannabinoids compared to all other parts of the plant [49].
Interestingly, even though climate intensities were lower in Pakistan, Pakistani HLE showed higher activity against Gram-negative bacteria [42]. According to Walsh et al. (2003), the potency of antimicrobial effects of hemp products depends on the type of seed and the part of the plant being used but not by the climatic indicators or soil, as had once been assumed [50].
India, Serbia, Kazakhstan, and Russia were all the sources of HEO, and all locations showed different antimicrobial effects. HEO sourced from India showed moderate to high antibacterial properties against Gram-positive bacteria and no activity against Gram-negative bacteria, apart from Salmonella typhimurium. Unlike the correlated results from Sudan, Pakistan, and Nigeria, HEO sourced from India obtained low antimicrobial effects despite the desirable climate between 9 and 39 °C in temperature, high rainfall between 6 and 260 mm, and humidity to 95%. The findings were due to the higher content of CBD in HLE compared to HEO. Both hemp plants grown in Pakistan and Nigeria were grown on garden soils and hemp plants in Sudan on woodland savanna/semi-arid soils, while Indian hemp was found on brown forest [31,42,43].
Serbian HEO showed lower antimicrobial activity. Its climate was significantly lower in all categories compared with Pakistan and Nigeria. HEO from Syrym Kazakhstan obtained the lowest temperatures, lowest rainfall, and lowest humidity levels throughout the year. Expectedly, it recorded the poorest antimicrobial activity, apart from Candida albicans, in Zheljazkov et al. [16]. These findings indicate that all three climate categories play a role in the antimicrobial activity of hemp plants and their oils. The three different HEO were sourced from Volgograd Oblast, Russia, with temperatures ranging from −10 to 30 °C, rainfall of 6-25 mm, and humidity of 0-2%. The rainfall and the temperature obtained are within the favorable climate conditions needed for hemp growth. Adesina et al. [3] reported that hemp grows best within a temperature range of 16 °C and 27 °C. The findings indicated that climatic conditions are not the only contributor to hemp oil's antibacterial and antifungal activity [3].

Soil Type on the Effects of Antimicrobial Activity of Hemp Oils
The hemp plant can be grown on several types of soil. However, it seems to favour loose, deep, well-drained loam soils rich in organic matter with an optimum pH between 6.0 and 7.5 [51]. Soil preparation for hemp is similar to that of barley or spring wheat. For seed production, hemp is harvested when seeds begin to shatter [51,52]. Hemp plants grown in gardens seem to exhibit the best antimicrobial activity in Pakistan and Nigeria. The possible reason may be assigned to the daily maintenance of gardens: watering, pesticide control, soil amendment, and weed control. The agronomic performance influences the quality and quantity of the bioactive compounds, as reported by Adesina et al. [3].
HSO from Sudan showed high to moderate antibacterial activity, especially against Staphylococcus aureus and other Gram-positive bacteria. Sudan lies on a woodland savanna with semi-desert soils, which are also known as semi-arid soils. Semi-arid soils contain deep, dark, sandy clay, and loam and are mostly dry all growing season. In addition, semiarid soils exhibit more organic matter whenever there is frequent rainfall and irrigation to supplement the rainfall deficit, as reported by Ros et al. [53]. Therefore, mostly in the semi-arid zones, hemp plants are usually grown under irrigated agriculture to increase the nutrient uptake for metabolic activities.
Chernozemic soils are described as black earth soil with high water-holding capacity that are calcium binding. They are humus-rich grassland soils with a pH of 7.2 and are commonly used for crop cultivation, including hemp plants in Backi Petrovac, Serbia [54]. HEO from Serbia possessed moderate antimicrobial activity; however, it was not as high as Russian 'Buro' and 'Saykaj' HEO sourced from Volgograd Oblast, Russia, with chernozemic and chestnut soils [16,30]. These two locations showed the highest antimicrobial activity against Gram-positive and Gram-negative bacteria in this study. They were both located on farmlands on opposite sides of the same river Volga. Both coordinates lead to a location near a small lake or swamp. 'Titelski Greg' HEO gave one of the poorest antimicrobial activities compared to other locations in this study. It was located in the same district as Saykaj, Mikhaylovsky District; however, it was not located near a lake, pond, or water source. The findings are clear indications that achieving the hemp plant water requirement will result in high antibacterial activity.
Humid soils located in Akzhaik and Syrym, Kazakhstan show moderate to low antimicrobial activity. Humid soils are deep and strongly weathered soils developed under aggressive, warm, and moist climates with high temperatures [55]. If the soil is too humid, it could promote pests, mold growth, and unwanted bacteria, altering the development of the hemp plant.
Lastly, hemp plants grown on forest soils or tarai soils showed moderate activity against Gram-positive and Gram-negative bacteria with little inhibition against fungi. These soils can either be rich with moisture from Artesan waters and swamps or drained from natural soil drains [56]. If the soil drains too rapidly, it will result in low water use efficiency of the hemp plant, as Larum (2020) reported [57]. According to Verma et al. [31], hemp plants cultivated on well-drained soil produce more antimicrobial properties, and HEO only had moderate activity against bacteria [31].

The Effects of Extraction Methods on Antimicrobial Effects of Hemp Oils
Three different extraction methods were compared to identify which extraction method gives the best antimicrobial effects in the oil: solvent extraction, cold pressing, and hydrodistillation; see Table 2. Out of the three types of solvent extractions used in all eleven experiments, solvent extraction using methanol exhibited the most robust antimicrobial inhibition of 40 mm ZGI against fungi Candida albicans [43]. HSO in Ali et al. [9] showed the second greatest ZGI of 28 mm against Staphylococcus aureus [9]. Green: G+ bacteria; Yellow: G− bacteria; Purple: Fungi Antimicrobial Activity; ZGI (mm), MIC (MICμg/mL), Decrease in viable bacteria (%) * no effect on Aspergillus niger. Units in (mm). A: Acetone. M: Methanol. All the abbreviations of the microorganisms are indicated after the conclusion.
Mkpenie et al. [43] observed if the type of solvent used to extract the HSO, acetone or methanol, affected the antimicrobial properties of HSO. The ZGI of fungi showed 5-12 mm less in acetone than methanol extraction. The difference in acetone and methanol extraction against Gram-positive and Gram-negative bacteria did not show any significant difference. Unrefined HSO was also extracted via methanol [4] and did not exhibit the same high result [9,42,43]. In addition, it had only 3 mm inhibition against Staphylococcus aureus, further confirming that the extraction method does not significantly influence antimicrobial events.
It is important to note that the temperature, rate of agitation, extraction time, and solvent to plant ratio all affect the final yield and antimicrobial potential of HSO [18]. Solvent extractions can be completed by a Soxhlet extractor or a DM technique [18]. However, ethanol seems to be the most popular choice of solvent used for extraction because it is considered adequate, efficient, and safe to handle [58,59], and methanol is better for the extraction of phytochemicals due to its polarity work [60].
Cold pressing as an extraction method has been conducted as a commonly used method due to its capacity to extract more phenolic compounds, chlorophyll, beta-carotene, flavonoids, gamma-tocopherols, alpha-tocopherols, and increase antioxidant capacity compared to solvent extractions [61,62]. However, the only disadvantage with cold pressing is that 60-80% of oil is out of the hemp seed [18 Fathordoobady]. Nevertheless, one study used a cold-pressing technique for HSO extraction and achieved an 85.7-90.7% decrease in viable bacteria against Staphylococcus aureus [41], suggesting promising results for cold-pressed HSO as an antimicrobial solution.
For traditional hydrodistilled methods, HEOs indicated moderate activity against bacteria and fungi [16,30,31]. There are three types of hydrodistillation techniques: water immersion, vapor immersion, and direct vapor immersion. The antimicrobial activity and major constituent extracted from the hydrodistilled oil are based on its Distillation Time (DT) and whether or not the hemp plant is grounded [30]. Hydrodistilled HEO showed promising results in CBD extractions of wild hemp plants and even pertained to a higher concentration of commercial hemp (7.5-7.8%) grown for CBD oil production [16]. Zheljazkov et al. [16] achieved up to 52.4% of CBD using hydrodistillation extraction, which was the highest recorded CBD content in a hemp plant using this extraction method, giving hydrodistillation a promising outlook for future tests [16]. Tables 3 and 4 show the inhibition zones of HSO, HEO, cannabinoids, and HLE against Gram-positive bacteria, Gram-negative bacteria, and fungi. * Zone of Growth Inhibition was not given, 5 mm was used as an estimate. Paper 2 showed some yeast inhibition during the first screening [32]. Units in (mm). n/a n/a n/a 7 [31] HEO was extracted from leaves, flowers, seeds, seed bracts, inflorescences, and thinner stems. Units in (mm).

HSO
HSO showed the highest activity against Gram-positive bacteria, moderate activity against Gram-negative bacteria, and little to no activity against fungi ( Table 3). The most sensitive bacteria to HSO was found to be Staphylococcus aureus, Micrococcus luteus followed by Bacillus subtilis. Ali et al. [9] showed the highest ZGI against Staphylococcus aureus out of all hemp-derived oils with an inhibition of 28 mm from the Cannabis seed oil itself and 12 mm in the whole plant. Inhibition of the seed oil was 8-16 mm greater than the gentamicin control and 16 mm greater than the whole plant [9]. The findings indicate that the content in the seed contains significant antibacterial effects against Staphylococcus aureus that are not found in the remainder of the plant. As a result of the effect of HSO on Staphylococcus aureus, HSO products can inhibit mainly skin infections such as acne and cellulitis, pneumonia, and bacteraemia. A likely reason HSO acts more deficient against Gram-negative bacteria is the outer protective lipopolysaccharide layer in the cell wall found in Gram-negative bacteria.
Nonetheless, HSO still expressed some inhibition against Gram-negative bacteria; therefore, it is a valuable oil to protect against bacteraemia, urinary tract infection, and pneumonia, often caused by Escherichia coli and Pseudomonas aeruginosa. However, more research should be conducted on HSO's ability to inhibit Salmonella enteritidis, Serratia marcescens, and Micrococcus luteus, since they gave higher inhibition rates in Mikulcová et al. [4] but were not tested against in Ali et al. [9]. In addition, HSO has been seen to inhibit yeast; however, it failed to replicate the results [32 Leizer].

HEO
HEO showed low to moderate activity against all bacteria and fungi. Staphylococcus aureus was the most sensitive Gram-positive bacteria to HEO and Pneumococcal pneumoniae and Streptococcus mutans. Staphylococcus aureus was most sensitive against the 'wild Buro' HEO, containing 52.4% of CBD (Table 4). The other three Gram-positive bacteria experienced some but little inhibition, starting with the highest ZGI against Pneumococcal pneumoniae, Enterococcus faecalis, and Bacillus subtilis. Interestingly, HSO expressed high activity against Bacillus subtilis compared to HEO. However, HEO showed higher activity against fungi. Due to the HEO content, various parts of the plant may potentially exude antifungal properties from compounds found in different parts of the plant. The HEO use exhibited the highest antimicrobial activity against Candida tropicalis once grounded and distilled for 5-10 min, which was followed by Candida albicans and Candida krusei, which was not grounded at 0-5 DT. Candida albicans was also most sensitive to the 'wild Saykaj' oil, which is grown in Russia. In general, HEO showed lower antifungal properties in each control. Fluconazole's inhibition against fungi averaged 3.5 to 15.7 mm higher than HEO in both papers. All bacterial inhibitions were lower than that of the control, suggesting that HEO is not an overly strong antimicrobial product. Table 5 shows that cannabinoids, especially CBD, THC, and CBN, extracted from flowers showed lower MIC values to Gram-positive Staphylococcus aureus and Staphylococcus epidermidis, which means that cannabinoids do not need to be present in high concentrations to exhibit antimicrobial effects. It has been reported that even the smallest amount of CBD or Cannabidiolic acid (CBDA) contamination in oil can positively impact Grampositive bacterial inhibition [16,32]. Since the flower containing all the beneficial cannabinoids is located beside the seed, the flower could likely contaminate HSO during extraction, and this may explain why Ali et al. [9] obtained a significantly greater inhibition effect against Gram-positive bacteria than Mikulcová et al. [4] and Leizer et al. [32]. Contrastingly, unlike Gram-positive bacteria, neither CBD nor CBDA showed any activity against Gram-negative bacteria, Pseudomonas aeruginosa or Escherichia coli, as reported by Martinenghi et al. [13], showing that cannabinoids work best against Gram-positive bacteria, and other major constituents in the oil cause inhibition against bacteria or fungi in HSO.  [13,15]. Papers 10, 11 represent HLEs [42,43]. Units in (mm).

Cannabinoids and HLEs
Next to cannabinoids, Table 5 shows HLEs, which also are a large source of cannabinoids such as CBD [63,64]. Once again, HLE showed high activity against Staphylococcus aureus; however, it possessed higher inhibition rates against Gram-negative bacteria and fungi. Inhibition of Staphylococcus aureus was similar but slightly higher than HEO; however, it was not as high as HSO inhibition, ranging from 10 to 20 mm ZGI [8,42,43,].
Compared to HSO and HEO, HLE experienced one of the highest inhibitory zones against Gram-negative bacteria, ranging from 10 mm to 25.3 mm ZGI. Mkpenie et al. [43] showed similar results for both Gram-positive and Gram-negative bacteria [43]. However, Naveed et al. [42] displayed 12-15 mm higher inhibitory effects against Gram-negative bacteria [42]. Furthermore, this study showed the highest amount of activity against Escherichia coli and Pseudomonas aeruginosa. According to Audu Sambo et al. [65], Cannabis sativa leaves contain larger quantities of amino acids, which would explain the higher antibacterial activity of HLE, since amino acids exhibit high antimicrobial effects.
Lastly, fungi experienced the greatest sensitivity to HLEs. It showed 20-40 mm ZGI against Candida albicans; however, it showed no activity against Aspergillus niger. This result would suggest why HEO expressed higher antifungal properties to HSO, as its ingredients include hemp leaves, unlike HSO. This was the highest ZGI overall, suggesting that the HLE is a good supplement to consume if suffering from Candidiasis. This study further proves that HSO is primarily effective against the inhibition of Gram-positive bacteria and that the antifungal properties of the plant are situated in other parts of the hemp plant, i.e., the leaf.

Antioxidant Activity of Hemp Products
Many diseases are associated with the accumulation of free radicals. Therefore, to test for antioxidant activity, a plant can be tested for its scavenging activity against free radicals. DPPH, ABTS, and others are widely used as stable free radicals to observe the plant's scavenging abilities. All the papers tested for radical scavenging abilities against DPPH are presented in Table 6. Despite HSO's promising antioxidant components (γ-to-copherols, terpenes, and phenolic compounds), HSO still exhibited the lowest DPPH scavenging capabilities in three out of four research articles. HSO showed 0.43 μmol Trolox/mg less scavenging ability to sunflower seeds [20 Senila], 49.7% less DPPH scavenging activity ascorbic acid, and 5.0-9.32% less DPPH activity compared with the male Wistar rats fed on a standard diet in vivo. HSO [10 Uluata] was the only one that did not show the least amount of DPPH scavenging ability compared to other oils. This coldpressed HSO showed the second-highest scavenging ability, which was greater than that of terebinth, radish, stinging nettle, and laurel oil. HSO does not show a significant antioxidant ability because HSO is highly unstable because of the high content of unsaturated fatty acids [10 Uluata]. HSH, HSF, and HPH expressed slightly higher antioxidant activities based on their DPPH scavenging capabilities and may be assigned to the additional components found in the seed coat, resulting in higher antioxidant capacity. Chen et al. (2012) determined the IC50 values of HSH and compared them to the HSK (seed without the hull) [67]. They concluded that HSH achieved a 0.92/0.42 lower IC50 value than HSK, meaning that lower amounts of HSH will give the same antioxidant effect as HSH. Chen et al. (2012) further isolated two compounds that showed the most antioxidant activity: N-trans-caffeoyltyramine and cannabisin B [67]. Other studies have supported that these constituents pos-sessed antioxidant abilities [71][72][73]. Furthermore, HSF also showed potential in antioxidant capabilities, because HSF is made from the shelled defatted hemp seed or the hull. It is already known from the previous study that HSH contains larger amounts of antioxidant components, making HSF a great additive for baked goods as a source of antioxidants.
HPH is a soluble form of hemp protein and is a hydrolyzed hemp protein isolate from defatted hemp flour. It is often used in skincare and as a dietary protein powder. Girgih et al. [69,70] conducted in vitro experiments on HPH to test their scavenging abilities against DPPH. The results found that HPH performed better against DPPH in the later study and showed great potential as an antioxidant. Girgih et al. [70] also conducted an in vivo experiment involving Hyper-Sensitive Rats (HSR) that were fed four different diets and showed that lipid peroxidation levels decreased 20-week-old SHRs (T2) when compared to the young SHRs (T1) [70]. The presence of HPH in the diets led to a significant (p < 0.05) increase in plasma SOD and CAT levels in the T1 and T2 groups and a decrease in TPx levels. According to Girgih et al. [70], HPH contained antioxidant peptides that reduced the rate of lipid peroxidation in SHRs with enhanced antioxidant enzyme levels and total antioxidant capacity [70].
To conclude the antioxidant activity in hemp seeds, the HSH around the kernel possessed the most antioxidant potential compared to HSO, which only uses the dehulled hemp seeds for oil production. The results suggest that to achieve optimal antioxidant activity from the hemp seed, shelled hemp seed products such as HSF, HPH, or even hemp protein isolate (protein isolated from the hemp seeds) are needed.
Different parts of the plant in addition to HSO were utilized in this peer review, such as HSH, HPH, and HSF, due to the lack of papers focusing on the antioxidant properties of HSO alone. More studies should be conducted on the influence of antioxidant activity in HSO using the same method of testing and a variety of different sources of hemp plants.

Conclusions
In this study, HSO exhibits antimicrobial activity based on its major constituents (cannabidiol (CBD), β-caryophyllene, and limonene). The growing factors influenced the bioactivity of the HSO such that humid soils showed low antimicrobial activity; in contrast, semi-arid soils revealed high antimicrobial activity against skin infections endocarditis and osteomyelitis caused by Staphylococcus aureus. HSO showed the highest activity against Gram-positive bacteria, specifically Staphylococcus aureus, Micrococcus luteus, and Bacillus subtilis, but it was not limited to Gram-negative bacteria of Escherichia coli and Pseudomonas aeruginosa. In addition, three extraction methods (cold pressed, hydrodistillation, solvent extraction) are commonly used to extract HSO. However, the extraction methods have less influence on the HSO bioactivity. HSO worked best on Gram-positive bacteria, particularly Staphylococcus aureus, but other hemp-derived products with more CBD concentration such as Hemp Leaf Extract (HLE) performed better against fungi, particularly Candida albicans. In regard to the plant's growing conditions (temperature, humidity, rainfall), temperature exhibited the highest effect on inhibition levels against Gram-positive bacteria, followed by humidity and rainfall. HSO shows the greatest antibacterial ability against Gram-positive bacteria when grown in warm climates ranging from 20 to 39 °C. Furthermore, well-drained loam soils rich in organic matter seem to stimulate the HSO bioactivity. HSO did not show significant antioxidant activity; however, HSH, HSF, and HPH expressed promising DPPH scavenging ability. In conclusion, HSO can be used in research for its antimicrobial purposes against Gram-positive bacteria. More research is needed to elucidate how limiting factors influence the bioactivity of hemp-derived product oils.