Plant Natural Sources of the Endocannabinoid (E)-β-Caryophyllene: A Systematic Quantitative Analysis of Published Literature

(E)-β-caryophyllene (BCP) is a natural sesquiterpene hydrocarbon present in hundreds of plant species. BCP possesses several important pharmacological activities, ranging from pain treatment to neurological and metabolic disorders. These are mainly due to its ability to interact with the cannabinoid receptor 2 (CB2) and the complete lack of interaction with the brain CB1. A systematic analysis of plant species with essential oils containing a BCP percentage > 10% provided almost 300 entries with species belonging to 51 families. The essential oils were found to be extracted from 13 plant parts and samples originated from 56 countries worldwide. Statistical analyses included the evaluation of variability in BCP% and yield% as well as the statistical linkage between families, plant parts and countries of origin by cluster analysis. Identified species were also grouped according to their presence in the Belfrit list. The survey evidences the importance of essential oil yield evaluation in support of the chemical analysis. The results provide a comprehensive picture of the species with the highest BCP and yield percentages.


Introduction
The endogenous cannabinoid system (ECS) plays an important role in the immune response to an infection. At present, two cannabinoid (CB) receptors are described: cannabinoid type 1 receptor (CB1) and cannabinoid type 2 receptor (CB2), both G-protein coupled receptors [1]. The CB2 receptor represents the peripheral CB, due to its expression on circulating immune cells. However, studies have also found CB2 expression in the brain, such as cerebellum and microglial cells [2]. The CB2 receptor is involved in the attenuation of inflammatory immune responses. CB2 receptor pathway activation entails the suppression of cytokine release from immune cells and thereby dampening of the inflammatory response (immunosuppression) [3].
(E)-β-caryophyllene (BCP) is a bicyclic sesquiterpene hydrocarbon which is present in the essential oil of several plant species [4]. The Research Institute for Fragrance Materials (RIFM) evaluated BCP safety and the molecule has been approved by the Food and Drug Administration and by the European Food Safety Authority as a flavoring agent, which can be used in cosmetic and food additives [5]. Reports on oral sub-chronic toxicity support the safety of BCP for its proposed use also in medical food products [5]. BCP has been reported to be active against several disorders, with particular reference to cancer, chronic pain and inflammation [2]. Non-clinical BCP toxicity and an absence of adverse effects have been described [6]. Moreover, BCP can act as a selective agonist of CB2 [1], it activates peroxisome proliferator-activated receptor-α (PPAR α) [7] and has been recently involved in the prevention of

Results and Discussion
The database search (performed in July 2020) for the term caryophyllene provided 5867 entries. The search was then refined by selecting all papers with a chemical composition description. This selection provided 2604 entries, which were individually analyzed in order to select papers providing information on BCP percentage > 10%. Papers were then analyzed and the species binomial name, the plant family, the country of origin of samples and the plant part extracted were reported along with the BCP percentage and yield percentage. The total number of selected species was 295 (Table 1). Table 1 also lists the presence of the species in the Belfrit list [11].
In general, the 295 species belonged to 51 families and were reported from 56 countries worldwide. The essential oil containing BCP was extracted from 13 different plant parts. Out of 295 species, 34 were found to be listed in the Belfrit list, whereas for 51 species no data were available on the yield percentage. In many cases, the researchers used a small amount of plant parts (ranging from a few g to 200-300 g) from which it was impossible to evaluate the oil yield. However, in the majority of the other cases the yield was provided and hence reported (Table 1). The essential oil yield of 243 species ranged from 0.001 to 8.58%, whereas the BCP percentage of all selected species ranged from 9.8 (the threshold minimum level for species selection) to 75.6% (Table 2), providing an average percentage of 0.42% for yield and 27.4% for BCP. As shown in Table 2, variability was higher for yield percentages than for BCP percentage. The reason for the yield and BCP variability depends on several factors, including plant part, the quantity of plant material distilled and, most of all, the genetic variability and phenotypic plasticity of plants [303][304][305][306]. In order to look for plant species with the highest BCP and yield percentages, a scatter plot was obtained, as depicted in Figure 1. The highest yield and BCP percentages were found for Copaifera langsdorffii. High BCP percentages but with decreasing yields were found for Bursera microphylla, Scutellaria havanensis and Pentadesma butyracea. Copaifera species, popularly known as copaiba oil, are widely used in Brazilian popular medicine and the genus is known for its high essential oil yield and BCP content [135,307,308]. The genus Bursera belongs to the plant family Burseraceae and contains several aromatic spices producing oleo-gum resins, such as the traditional incenses, frankincense and myrrh [309]. Pentadesma butyracea (Clusiaceae) is a dense forest species which is found in the center and north of Benin forests whose bark, rough and deeply cracked, exudes a thick resinous juice, of reddish yellow color [115]. The Scutellaria genus (Lamiaceae) consists of plants which are widely distributed throughout the world; S. butyracea is an endemic plant native from Havana and is ethnomedically used for several purposes because of its BCP content [196].
The plant part that contained the highest content of BCP was then analyzed. In order to evidence the statistical linkage between the plant parts, a cluster analysis was calculated by considering as category the plant part and as variables the number of species, the BCP% and the yield% reported in Table 1 (Figure 2). Euclidean distances were calculated by using the average linkage method. Five clusters were evidenced: the first cluster was made by plant parts reported in more than 100 species and was dominated by leaves and aerial parts, which contained in general a BCP percentage lower than 28%. The other four clusters were made by plant parts reported in less than 16 species. These four clusters were further subdivided according to their BCP content ( Figure 2). As expected, the highest BCP percentage was found in oleo-gum resins (cluster 2), followed by roots, barks and branches (cluster 3). Flowers and buds (cluster 4) showed a high yield, whereas twigs and woods (cluster 5) had both low yields and BCP percentages (Figure 2).    The next analysis was at the familial level. A cluster analysis was calculated with average linkage method by using data of Table 1 by considering as a category the plant families and the species number, yield% and BCP% as variables. The results of the cluster analysis show the presence of 6 clusters ( Figure 3). The first cluster is made by the Asteraceae and the Lamiaceae which consist of a number of species > 50 and a BCP% < 31. The second cluster gathers all families whose species have a BCP% > 35%; in this cluster, the Magnoliaceae and the Papilionaceae are separated in a subcluster because of their high BCP% and low yield%, whereas the Fabaceae (which include the above mentioned C. langsdorffii) are separated in a subcluster because of their high yield %. The third cluster is made by families with a number of species > 13 and a BCP% > 23%; here, the Lauraceae, the Apiaceae and the Zingiberaceae are separated in a subcluster because of their higher BCP%. The genus Ocotea is one of the largest of the Lauraceae family, with approximately 350 species distributed throughout tropical and subtropical America. O. splendens, as many other Ocotea species [212] is characterized by a high percentage of BCP [217]. In the Apiaceae family, the species P. kotschyana spreads widely through Anatoly, Iran (northwest, west and center) and north of Iraq and contains BCP in all plant parts [41]. The family Zingiberaceae is well known for producing essential oils that are used to prevent and control several diseases; the species R. breviscapa was found to possess a high percentage of BCP [300]. The fourth cluster is made by families with a BCP% > 26 and a subcluster separates the Atherospermaceae, the Flacourtiaceae and the Meliaceae because of their BCP%. The fifth cluster is made by families with a BCP% < 25 and the Plantaginaceae are separated in a subcluster because of their relatively higher yield%. Finally, the sixth cluster is made by plant families with a low BCP percentage and a subcluster separates the Hernandiaceae, the Juglandaceae, the Phyllanthace and the Ptaeroxylaceae because of their BCP content lower than 11%.  Table 4 describes the statistical data related to plant families. The next analysis aimed to evidence the geographical areas from which the plant species listed in Table 1 were collected. A cluster analysis was calculated with average linkage method, considering the country of origin as a category of their species number, yield% and BCP% as variables. The results of the cluster analysis show the presence of 6 clusters ( Figure 4). The first cluster gathers countries with the highest number of species and a BCP percentage higher than 28%; here, a subcluster separates Brazil from India and Iran because of the higher number of species, in agreement with the literature data [310]. The second and third clusters identify countries where BCP has the highest percentages, whereas the fourth cluster gathers countries with a number of species higher than 8. The fifth cluster is made by countries where the BCP content is the lowest, whereas the sixth cluster is made by two subclusters with BCP percentages ranging from 18 to 25%. One of these subclusters is made by countries (Colombia, Fiji, Kenya, Morocco, Niger, North Korea, Portugal and Togo) where the species had a BCP percentage higher than 24% (Figure 4).   Table 5 summarizes the statistics related to countries of origin. In order to separate which species containing BCP were also represent in the Belfrit list, a scatter plot was obtained by selecting BCP% and yield% as variables ( Figure 5). C. langdorffii, S. aromaticum, C. longa and B. virgilioides were characterized by a yield ranging from 2 to 28%, with varying percentages of BCP; on the other hand, high percentages of BCP but lower yields% were found for A. eupatoria, H. coronarium, C. odorata, P. americana and M. keonigi. All other species showed both lower yields and BCP percentage.

Systematic Analysis of BCP-Containing Plant Species
After a preliminary search by using different databases, the work was performed by using Clarivate Analytics Web of Science as a database (http://apps.webofknowledge.com). The basic search criterion was on the general search for the molecule (caryophyllene), then the exclusion criteria were the presence of BCP and a percentage of BCP in the reported results higher than 10%. Papers reporting the occurrence of BCP where then downloaded and saved as a pdf for further reading and collection of information.

Statistical Analysis
The binomial name of the species (including the author), the family of belonging, the plant part used, the country of origin of the sample, the yield and the BCP percentages were inserted in a database by using Systat ® 10 software (Systat Software Inc., San Jose, California, U.S.A.). Data were organized in columns and used for further processing. Average values along with ranges, standard deviation (S.D.), standard error of the mean (S.E.M.) and coefficient of variation (C.V.) were calculated by considering as grouping categories either the species, families, country of origin or plant part used.
As a classification statistical method, a cluster analysis was calculated by considering for each category the total number of species, the BCP percentage and the yield percentage by using Systat ® 10 software. Euclidean distances were calculated with the average linkage method. Data were plotted as either scatter plots of yield percentage vs. BCP percentage or dendrograms showing the different clusters according to the calculated distance.

Conclusions
The attractiveness of BCP, a natural sesquiterpene present in the essential oil of different plant species, arises from its pharmacological feature as a CB2 receptor agonist. This characteristic, along with the lack of interaction with the CB1, makes BCP an interesting plant endocannabinoid with the advantage of lacking any psychotropic effect, as is typical of some Cannabis extracts [8,311,312].
This systematic analysis of published literature on plant species containing BCP in their essential oils identified the species with the highest yield and BCP content and allowed to select which species are also present in the Belfrit list (i.e., potentially attractive for pharmaceutical and nutraceutical industries).
This survey also evidenced the common practice of many authors to ignore the importance of providing the yield of the distilled essential oil, which represent a basic starting point for all industrial applications of the plant species under study. This problem was often correlated with the low amount of plant material distilled. Although interesting from a chemical-analytical point of view, the sole chemical analysis of the essential oil is not useful if performed on a single plant or a few plants, because it does not provide any information on the population genetic variability, being mainly affected by phenotypic plasticity, which is responsible for individual variations inside a population [305].
This work identified some top species like C. langsdforffii, C. odorata, H. lupulus, P. nigrum and S. aromaticum, which provide a high percentage of BCP along with interesting yields. These species, upon a skillful molecular fractionation to remove undesired/toxic monoterpenes, may provide high percentages of BCP that can be used for the preparation of new drugs or dietary supplements aimed to improve health, prevent lifestyle diseases and act as a valid support for chronical diseases such as pain, metabolic and neurological disorders.