Chemical and Biological Studies of Cannabis sativa Roots

The chemical study of Cannabis sativa roots led to the isolation and identification of 10 compounds. Their chemical structures were unambiguously established on the basis of 1D and 2D NMR spectroscopy and mass spectrometry as friedelan-3-one (1), epifriedelanol (2), β-sitosterol (3), ergost-5-en-3-ol (4), methyl hexadecanoate (5), pentadecanoic acid (6), 10E-hexadecenoic acid (7), 4-hydroxy-3-methoxybenzaldehyde (8), β-sitosterol-β-D-glucoside (9) and p-coumaroyltyramine (10). Compounds 5–9 were reported for the first time from C. sativa roots. All the isolated compounds were tested for their antimicrobial activity. Compound 4 showed modest activity against Cryptococcus neoformans with an IC50 value of 13.7 μg/mL, while compound 10 displayed potent activity against Escherichia coli with an IC50 value of 0.8 μg/mL. A high-performance liquid chromatography method was developed and validated for the detection and quantification of p-coumaroyltyramine (10) in the extracts of different varieties of C. sativa roots.


Introduction
Cannabis sativa L. is one of the most widely used plants for both recreational and medicinal purposes. To date, a total of 567 natural constituents covering several chemical classes have been identified from C. sativa [1,2]. The most important classes are the cannabinoids, terpenoids, nitrogenous compounds, noncannabinoid phenols, flavonoids, and steroids [3]. The principal use of cannabis in medicine is for easing pain and in ameliorating nervous system disorders. It is reported to be useful in the treatment of gout, neuralgia, rheumatism, insanity, and insomnia among others, with actions almost entirely on the higher nerve centers [4].
The first reference to cannabis consumption dates as far back as 2,700 BC in China, with Shennong pên Ts'aoching, one of the oldest Chinese medicine books, mentioning the use of cannabis roots as a remedy to sooth pain. Throughout history, cannabis roots were documented in ancient Greek medicine, and a medical article in how Indians boiled them together with other leaves to make poultices for the treatment of inflamed skin surfaces and skin rash [3,5]. There are numerous reports on the traditional use of cannabis root for the treatment of fever, inflammation, gout, arthritis, and joint pain, as well as skin burns and hard tumors [3]. Also, they were used to treat postpartum hemorrhage, difficult child labor, sexually transmitted disease, and gastrointestinal activity and infection [6]. Despite a long history of therapeutic use, the roots of cannabis plants have been ignored in modern medical research and practice. Cannabis roots have been reported to have many different compounds, including triterpenoids, monoterpenes, alkaloids, sterols, amides, and choline [3].
On continuation of our search for bioactive compounds from C. sativa [7,8], this article describes the isolation and structural elucidation as well as the antimicrobial activity of 10 compounds from C. sativa roots. A high-performance liquid chromatography (HPLC) method was also developed and validated for the quantification of p-coumaroyltyramine (10) in extracts of different varieties of C. sativa roots which could be used as a possible marker compound to distinguish between roots of different varieties of C. sativa.

General Experimental Procedures
1D and 2D NMR spectra were recorded using the residual solvent signal as an internal standard on Bruker BioSpin Gm bH 400 and 500 NMR spectrometers (Bruker, Rheinstetten, Germany). Thin-layer chromatography (TLC) was carried out on aluminum-packed plates precoated with silica gel F 254 (Silicycle, Quebec, QC, Canada). Visualization was accomplished by spraying with a vanillin sulfuric acid spray reagent followed by heating.

Plant Material
C. sativa plants were grown at the University of Mississippi, USA and identified by Dr.
Suman Chandra, senior scientist, NCNPR, School of Pharmacy, University of Mississippi. The fresh roots were washed with tap water followed by distilled water and dried under shade. The dried roots were powdered using a coffee grinder.

In vitro Antimicrobial Assay
All organisms used for the biological evaluation were obtained from the American Type Culture Collection (Manassas, VA, USA). These include the fungi Candida albicans ATCC 90028, Cryptococcus neoformans ATCC 90113, and Aspergillus fumigatus ATCC 90906 and the bacteria methicillin-resistant Staphylococcus aureus ATCC 43300 (MRS), Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 25955, and vancomycin-resistant enterococcus. Susceptibility testing was performed using a modified version of the CLSI (formerly NCCLS) methods as previously described [9,10].

HPLC Analysis of p-Coumaroyltyramine (10)
Reagents and Materials-Acetonitrile (CH 3 CN), MeOH, tetrahydrofuran, and H 2 O are of HPLC grade (Fisher Scientific, Fairlawn, NJ, USA). Compound 10 was isolated from the roots of C. sativa. The chemical structure of compound 10 was identified by 1 H NMR, 13 C NMR, heteronuclear multiple-quantum correlation, heteronuclear multiple-bond correlation, and electrospray ionization mass spectrometry. The 1 H and 13 C NMR data of compound 10 are shown in Table 1 and Figure 1. The purity of compound 10 was determined on the basis of UV, NMR, MS and HPLC to be >98%.
Apparatus and Chromatographic Conditions-A Waters 9526 HPLC system equipped with a quaternary solvent delivery system, an autosampler and a DAD detector were used. Separation was achieved on a Phenomenex luna C18 column (250 × 4.6. mm i.d., 5 μm particle size). The mobile phase consisted of (a) CH 3 CN:MeOl·l (1:1) and (b) H 2 O. The gradient elution (% a in b) was as follows: 0-8 min, linear gradient from 30 to 45%; 8-10 min, linear gradient to 50%; 10-15 min, linear gradient to 60%, which was held for 5 min then returned to 30%. Each run was followed by equilibration time of 5 min. The flow rate was 1.0 mL/min and the total run time was 20 min. The column temperature was set at 30° C. DAD spectra were monitored from 210 to 480 nm and the detection wavelength was set to λ max 290 nm. The injection volume was 10 μL. Method Validation-The method was validated for linearity, precision (interday, intraday and intermediate precision), accuracy, stability, specificity, and selectivity following the International Conference on Harmonization (ICH) guideline [11].

Antimicrobial Activity
The antimicrobial activities of all isolated compounds were determined against methicillinresistant S. aureus (MRSa), E. coli, P. aeruginosa, and Mycobacterium intra-cellulare, as well as against pathogenic fungi including C. albicans, A. fumigatus, and C. neoformans. Compounds 4 and 10 showed antimicrobial activity. Compound 4 was active against C. neoformans with an IC 50 value of 13.7 μg/mL, while compound 10 was active against E. coli with an IC50 value of 0.8 μg/mL ( Table 2).

HPLC Analysis
Plant constituents vary considerably based on several factors such as temperature, light, drying, packing, and storage, which may impair not only the quality of phyto-therapeutic agents but also their therapeutic value [20]. Thus, standardization of raw materials and herbal preparations needs to be permanently carried out in term of quality specification, stability profiles and chemical analysis of analytes of interest using sensitive validated analytical methods [21]. HPLC is a unique, versatile, universal and well-recognized tool for qualitative and quantita tive evaluation of herbal products against their respective bioactive molecules in terms of quality and batch-to-batch reproducibility [22]. Thus, in this study contribution, we have developed a simple, economic and rapid chromatographic method using RP-HPLC for the estimation of p-coumaroyltyramine (10) in different varieties of C. sativa roots.

Method Validation
Compound 10 was detected and quantified by HPLC, using a gradient mobile phase consisting ofCH 3 CN:MeOH (1:1) and H 2 O. Compound 10 showed a sharp peak at 8.81 ± 0.015 min under the optimized chromatographic conditions at λ max 290 nm. Representative chromatograms are depicted in Figure 3. The separation of the marker compound (10) in a short time enabled rapid analysis of the samples. The calibration curve showed good linearity relationship in the specified concentration range (1-100 μg/mL) with a correlation coefficient (r 2 ) of 0.9996 ( Fig. 3; Table 3). The limits of detection and quantification were found to be 0.025 μg/mL and 0.1 μg/mL, respectively, thus suggesting a high sensitivity of the method which can be successfully exploited for quantifying even low sample concentrations of compound 10 ( Table 3). The relative standard deviation for system suitability in terms of R t and area were found to be less than 6% indicating the stability of the chromatographic method. The percent relative standard deviation of inter-and intraday analysis of standard and extract were also found to be less than 7 with a high repeatability of both R t and response (Table 3). Mean recovery for the quality control samples of pcoumaroyltyramine (10) was found to be >98% ( Table 4). Because of the almost quantitative recovery of compound 10 and the consistency of the analysis, the external standard method was adopted for quantification.

Method Application
The validated method was employed for the quantitation of p-coumaroyltyramine (10) from different varieties of C. sativa roots, namely high CBD, intermediate and high THC varieties.
The HPLC profiles of the cannabis extracts samples showed a sharp peak for compound 10 at R t 8.81 (± 0.015) min comparable to the standard. Figure 4 demonstrates a clear baseline separation of compound 10 in the three varieties of cannabis from adjacent peaks. The content of compound 10 in the high CBD variety was 19.78 ± 0.728 μg/g, while in the intermediate and high THC varieties it was 8.00 ± 0.348 and 7.65 ± 0.359 μg/g, respectively. The representative chromatograms and values are shown in Figure 4 and Table 5, respectively.

Conclusion
Ten compounds have been isolated and identified from a high CBD variety of cannabis roots, of which compounds 4 and 10 showed promising antimicrobial activities. A validated HPLC method for the quantitation of compound 10 in three varieties of C. sativa was developed. The method was fast, simple and accurate and could be used for routine analysis of this marker compound in cannabis roots. Whether this compound can be used as a marker to discriminate between CBD variety roots and the other two varieties need to be further investigated.   In vitro antimicrobial activities of compound 4 and 10 (IC50 in μg/mL) LOD, limit of detection; LOQ, limit of quantitation; RSD, relative standard deviation.