Chemical profiles as chemotaxonomic tools for Loranthaceae in Nigeria

Department of Medicinal Plant Research and Traditional Medicine, National Institute for Pharmaceutical Research and Development, PMB 21, Garki, Abuja, Nigeria. Department of Pharmacy. University of Maiduguri, Maiduguri, Borno State, Nigeria. Department of Medicinal Chemistry and Quality Control, National Institute for Pharmaceutical Research and Development, PMB 21, Garki, Abuja, Nigeria. Department of Botany, University of Ibadan, Ibadan, Oyo State, Nigeria.


INTRODUCTION
Mistletoes are widespread throughout Africa, North America, Asia, Europe, Australia and Malaesia, with the American mistletoe (Phoradendron serotinum) and the European mistletoe (Viscum album) particularly well known.Different species growing on different hosts may synthesize toxic compounds and protein such as lectins *Corresponding author.E-mail: sadiqoyene@yahoo.com.Tel: +2348058293853.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License and alkaloids with varying pharmacological activities (Preston et al., 2010).Thus, both the mistletoe and its host have shared responsibility in determining the pharmacological activity of the species.The distributions of these compounds or metabolites in different parts of a plant also vary (Preston et al., 2010).These pharmacological effects are due to variation in the chemical profiles especially the profiles of secondary metabolites.Secondary metabolites are proteins, glycosides, phenolics, steroids, saponins, terpenes, alkaloids and other chemical substances.Takhtajan (1973) suggested that secondary metabolites are compounds that may have taxonomic relevance.
Eighty percent or more of the world's population is estimated to depend primarily on traditional medicine for the treatment of ailments (Cunningham, 1993), and as a matter of fact, the use of medicinal plants is the main means of treatment by traditional healers.Also, many useful compounds, which are today used for treatment of life threatening diseases, were isolated from medicinal plants e.g.Artemisinin from Artemisia annua L. and Vincristine from Catharantus roseus (L.) G. Don (Dana, 2012;Aslam et al., 2010).The Loranthaceae, a parasitic family with mistletoes members are often considered useful as medicinal plants.It has been documented that mistletoes have immeasurable medicinal and traditional uses (Burkil, 1995;Erturk et al., 2003).The biological activities of immunomodulatory and antitumor effect of some mistletoe may be attributed to the presence of metabolites like lectins, viscotoxins and alkaloids found in the parasites (Stirpe et al., 1982;Bussing et al., 1996;Fernandez et al., 1998;Stein et al., 1999;Mengs et al., 2002).
The wide array of secondary metabolites in Loranthaceae sp. is believed to be of chemotaxonomic importance.(Tilney and Lubke, 1974), and chlorogenic acid was found in all the species.Gedalovich-Shedletzky et al. (1989) analyzed and compared the chemical composition of viscin mucilage from three mistletoe species.Chemical analyses of different extracts from Agelanthsu dodoneifolius yielded components such as triterpenes, sterols, carotenoides, saponosides, anthracenosides, anthocyanosides and tannins (Traoré, 2000).However, chemotaxonomic information on the West African or Nigerian species is unavailable.To clarify the status of Loranthaceae in the region, a revision of the Nigerian species was carried out recently and about 15 species were documented for the region (Ibrahim and Ayodele, 2011).This study aimed to determine the profile of some basic secondary metabolites in the Nigerian species, which could be of chemotaxonomic significance.

MATERIALS AND METHODS
All reagents used were of analytical grade and were purchased from Zayo-Sigma Abuja, Nigeria.TLC plates used were also from the same source.

Plant collection and preparation
Twenty-seven specimens belonging to seven species were collected from the field through a field survey across host plant species and geographical location (Table 1).The specimens include Agelanthus dodoneifolius (4), Globimetula braunnii (4), Phragmanthera capitata (2), Phragmanthera nigritana (1), Tapinanthus bagwensis (4), Tapinanthus cordifolius (4) and Tapinanthus globiferus (8).Vouchers specimens were deposited at the University of Ibadan Herbarium (UIH) The leaves of each specimen were air-dried for one week at ambient temperature, and then pulverized using a mortar and pestle.The powdered leaf samples were used for the phytochemical screening and thin layer chromatographic (TLC) profiling.
Two grams of powdered leaf samples of each specimen were macerated in 20 ml of acetone for 24 h and filtered using filter papers.The extracts were spotted on three different pre-coated silica gel normal-phase TLC plates of dimension 12.5 by 8.5 cm.The dry spots were developed in a TLC tank of solvent system of ethylacetate : chloroform : methanol : water, in the ratio of 15:8:4:1.The developed spots were visualized by spraying the first plate with Vanillin in sulphuric acid reagent, the second plate with Gibbs reagent and the third plate with Dragendoff reagent for detection of terpenoids, phenolics and alkaloids, respectively.The retention factors (R F values) were calculated for all the spots as distance moved by spot from the origin divided by distance moved by solvent front (Table 2).

Phytochemical screening
The result of the phytochemical screening for secondary  Key: See Table 3.
dodoneifolius on P. biglobosa from Suleija and T. bangwensis on N. laevis and C. medica (Table 1).Each of the four specimens of T. bangwensis on different host plants from Ibadan lacked tannins and also the two specimens of G. braunii from Ibadan lacked tannin (Table 1).Phenolics were also found to occur in most of the specimens screened except G. braunii on Theobroma cacao and T. bangwensis on C. medica (Table 1).
Figure 1 shows percentage response of the specimens to the metabolites screened.From this study, none of the metabolites occurred in all specimens or even all species (Figures 1 and 2).
Figure 2 present the percentage of species responding to metabolites in each location while Figure 3 shows the percentage response to metabolites by the species.Generally, about 90% of the species tested positive for balsam and phenols, while 76% tested positive for tannins, 63% for saponins and less than 5% for phlobatannin (Figure 1).All the samples of G. braunnii tested positive to balsam and saponins, all P. capitata tested positive to balsams, flavonoids and tannins, P. nigritana tested positive to balsams, flavonoids, tannins, saponins, terpenes and phenols, while samples of T. bangwensis varied in their chemical profiles with no consistent positive indication for a particular metabolite.However, over 75% tested positive to balsams, flavonoids, and phenols, while about 75% also tested positive to glycosides, resins, balsams, volatile oils, saponins and terpenes.All samples of T. globiferus tested positive to balsam, flavonoids and tannins and 75% was positive to phenols.

TLC profiling
The TLC profiling of the specimens of    Loranthaceae sp. using Gibbs, vanillin-sulphuric acid and Dragenddoff spray reagents for TLC are shown in Figures 4, 5 and 6, respectively.In Figures 4, 5 and 6, Gibbs reagent were used for visualizing phenolics, vanillin in sulphuric acid for terpenoids, while Dragendoff reagent was used to see if alkaloids were present on the TLC plate, respectively.Table 2 shows the R f values of spots found on the TLC plate in Figure 4, which reveals that all the specimens had phenolics in them although to varying degree judging from the numbers of spots.T. cordifolius on Cassia sp. from Jos (spot 12), G. braunii on P. americana (spot17) and G. braunii (spot 18) from Calabar have the highest number of spots of 13, 13 and 12, respectively (Table 2).An intermediate number of spots were found in T. globiferus on P. curattelifolia from Kano (spot 4), P. nigritana on Citrus sp. from Suleija (spot 16) and G. braunii on Cola sp. from Ibadan (spot 19) with 10, 11 and 11 spots respectively (Table 2).The lowest spots are found in T. globiferus on A. indica (spot 2), T. globiferus on Tectona grandis (spot 3), T. globiferus on Zyzyphus sp.(spot 5), T. bangwensis on Newboldia laevis (spot 10) and Agelanthus dodoneifolius on P. biglobosa (spot 21) with 3, 5, 3, 3 and 5, respectively (Table 2).Spots with R F values of 0.29 -0.32 and 0.71 -0.73 are found to be present in over 90% of the specimens.In Figure 5, terpenoids were only observed in some of the specimens.Alkaloids were absent from all the specimens studied (Figure 6).

DISCUSSION
The phytochemical analysis and the TLC profiling showed variation in the constituent secondary metabolites among various species irrespective of their host and ecological location (Table 1; Figures 4 and 5).Variation in secondary metabolites among the same mistletoe species occurring on different host plants have been observed in earlier studies (Deeni and Sadiq, 2002;Ibrahim et al., 2009).The only consistent pattern from this study was the lack of alkaloids from all the specimens analyzed (Table 1; Figures 1, 2 and 6).It is a known fact that quantitative and qualitative information on secondary metabolites is useful for taxonomic classification of plants (Harborne, 1968;Takhtajan, 1973).Hence absence of alkaloids, and the number of species testing positive for balsam and phenols appears to be of chemotaxonomic significance among the species in Nigeria.Alkaloids were not recorded for any of the Nigerian Loranthaceae specimens studied but Sanchez-Areola et al. (2004) recorded the presence of alkaloids in Psittacanthus calyculatus, a New World Loranthaceae endemic to Mexico (Kuijt, 2009).
The TLC R f in Table 2 shows that there were similar phenolic compounds (R f values of 0.29 -0.32 and 0.71 -0.73) present in most of the specimens, over 90% of the species and this further reinforced the fact that phenolics could be a source of analytical marker compound(s) for standardization of herbal preparations from these species.High amounts of phenolics have long been known to be a phytochemical feature of parasitic flowering plants and they are said to occur at a level that is generally higher than the host plant (Khanna et al., 1968;Salatino et al., 1993).The study reveals that G. braunii specimens irrespective of their hosts or locations are rich in phenolic compounds as compared to other species while T. globiferus and T. bangwensis are depauperate in phenolics as compared to other species.Also of note is the absence of glycosides in the Phragmanthera species and total absence of tannins from all the specimens from Ibadan.These findings may be of chemotaxonomic importance.Thus, the presence of balsams and phenols could be used in specific combination with morphological characteristics and biogeographical distribution ranges for the delineation of genera and species in the family (Crockett and Robson, 2011).
Research on dwarf mistletoes (Viscaceae) in North America indicates that plant chemistry, particularly secondary metabolites, plays an important role in determining interactions between host and parasite (Snyder, 1996).This may not be applicable to Nigerian Loranthaceae because of the variation noted in the metabolites present in the same species on different hosts.Differences in chemical profiles of the various species studied underscore why the specific choice of species for the treatment of a particular ailment is very important.This study has shown that some species may not possess a particular metabolite that is common in other species.For instance, the absence of glycosides in Phragmanthera species or tannins and saponins in T. bangwensis may result in major pharmacological differences.Although the correlation between host and chemical profile of the species was not clearly defined in this study, it is believed that the host could play a role in the observed chemical profile of the plant or species.The influence of host chemistry on the chemical constituents of the parasite on different hosts might justify why the host is as important as the parasite in pharmacognosy, ethnopharmacology and ethnomedicine, and why the use of these Loranthaceae in the treatment of an ailment is often dependent on a particular or specific host (Burkill, 1995;Snyder 1996;Adodo, 2002;Olapade, 2002;Preston et al., 2010), for instance, in Brazil, where there is preference for Cladocolea micrantha growing on cashew tree (Anacardium occidentale) for the treatment of tumors and inflammatory diseases (Adodo, 2002;Olapade, 2002;Guimaraes et al., 2007).

Conclusion
From this investigation, species of Loranthaceae in Nigeria might not be delineated by scoring presence or absence of their secondary metabolites qualitatively or quantitatively due to variations which occur on same species form different hosts but the occurrence of similar metabolites like phenolics and balsam in most, if not all the species irrespective of the host and locality is useful taxonomically as a marker for the group.It is therefore our recommendation that caution should be exercised in the use of Loranthaceae as phytomedicine because of the chemical variations which exist in the same species found on different hosts.The same species collected from two different hosts might have different pharmacological effects in the body.The group is currently working on determining a phytochemical marker for the family Loranthaceae in Nigeria.

Table 1 .
Preliminary phytochemical screening of secondary metabolites from Loranthaceae species in Nigeria including taxa, hosts, localities, collection numbers and metabolites studied.
metabolites of species of Loranthaceae on different hosts from different localities is presented in Table1.Alkaloids were absent in all the species of Loranthaceae screened.Flavonoids were present in all except few specimens, A. dodoneifolius on Parkia biglobosa from Jos, the two specimens of G. braunii collected on an unidentified host from Calabar, P. capitata on Persea americana from Calabar and Tapinanthus bangwensis on Citrus medica from Ibadan (Table1).Balsams occurred in all the specimens of the Loranthaceae species except few species like A.

Table 2 .
R F values of phenolic spots from TLC profile of Loranthaceae species in Nigeria.

Table 3 .
List of specimens, hosts and their corresponding extract spot number on the TLC plates {(Figures4-6) and Table2}.