Mistletoe Contains Higher Secondary Metabolites Than the Host Plant at the Host-Parasite Interface: Insights From Tapinanthus Globiferus Collected In Enugu, Nigeria

Background: This study aims at evaluating the phytochemicals composition at the host-parasite interfaces of parasitic plant Tapinanthus globiferus (mistletoe) and four host plants. Wood tissues of the hosts and the parasite at the host-parasite interface were collected and analyzed to determine the presence secondary metabolites. Results: The result showed that avonoids, saponins, and glycosides were present in the plants and parasite samples. The results revealed higher concentration of avonoids (P < 0.05) in the parasite of C. acuminata (1190.33 ± 48.23 mgQE/g) and P. macrophylla (1482.55 ± 31.35 mgQE/g) than in the host plant. Saponins was signicantly (P < 0.05) higher in the parasites as compared to their respective host. Conclusion: At the host-parasite interface, signicantly higher phytochemicals in the wood portion of T. globiferus was observed as compared to the host plants wood; however, the variability in phytochemical content of T. globiferus is dependent on the host. Therefore, milestoe would be a better source of bioactive compounds with high medicinal values than their host plants if explored further.


Background
Enormous biologically active compounds with a variety of chemical structures and properties are all deposited in the plant kingdom. [1] Phytochemical is a broad word generally used to describes a wide diversity of compounds that are found naturally in plants. They are found in the different parts of the plants; such as roots, stem, barks, leaves, owers, seeds and pulps. [2] Phytochemicals otherwise known as secondary metabolites present in smaller quantities in higher plants, include the avonoids, alkaloids, terpenoids, tannins, steroids etc. [3] These bioactive secondary metabolites helps the plant to overcome temporal or continuous threats integral to their environment, while also controlling essential functions of growth and reproduction. In other words, they are essential for the survival and proper functioning of plants. [4] Medicinal properties of plants are dependent on a number of chemically active substances that produces a de nite physiological action on a biological assay. In general terms, the phytochemicals in plants play an important role in their medicinal properties. Nevertheless, medicinal plants are the richest bio-resource of drugs in traditional system of medicine and the phytochemicals are responsible for the different avours, colours, and smell of plants. These secondary metabolites (phytochemicals) have recently become of great interest owing to their versatility. [5] Phytochemicals in plants are been utilized globally as the traditional herbal medicine. These phytochemicals are present in the different plant parts and are used for healing of certain disorder like diabetes, arthritis, cancer, etc. [6] Secondary metabolites are economically vital in the manufacturing of drugs, fragrances, dyes, pesticides and food additives. Therefore, researchers have laid more emphasis on phytochemical studies. [2] Tapinanthus globiferus (mistletoes) are the predominant group of plant semi-parasitic ever green shrubs which belong to the family Loranthaceae. They grow on the branches of host trees or shrubs and take water and nutrients from the host's vasculature. Mistletoes include notorious parasites and are capable of destroying the trees and shrubs of economic value. However, they have been reported to attack a large number of varieties of taxonomically unrelated hosts and their attack has been proved to be fatal to various trees and shrubs. [7] This parasitic plant thrives on deciduous trees preferring those with soft bark like old apple trees, guava, cocoa, citrus and other trees.
Though Mistletoes are generally recognized as destructive agents to various valuable species, they do not seem to have received much attention from both researchers and plant scientists. Presence of various phytochemicals such as glycosides, alkaloids, viscotoxins, phenylpropannoids, tannins, lignins, lectins and sugars has been reported in the mistletoe collected from different host plant. Mistletoes found on various host trees is endowed with different antioxidant activity; however, the antioxidant capacity of the extract could vary according to the harvesting time of the plant as well as nature of the host tree [8,9] . Very often, host trees that are attacked by mistletoes suffer from them as the triumph of mistletoes lead to poor growth and productivity and eventual death of such host plants. The reason for the successful parasitism of mistletoes may be related to the phytochemical interaction between the host and the parasite. Nevertheless, there is insu cient information on the phytochemical study of parasitic plants and their hosts in order to detect the secondary metabolites present at the host-parasite interface. The increasing interest in powerful biological activity of phytochemicals outlined the necessity of determining their composition at the host-parasite interface of T. globiferus and its four named hosts. This study generally aims at evaluating the phytochemical composition at the host-parasite interfaces among Citrus sinensis, Pentaclethra macrophylla, Cola acuminata and Persea americana.

Results
The qualitative phytochemistry as presented in Table 1 shows that only avonoids, saponins and glycosides were present in the different plant host and parasites at different level of abundance. The Flavonoids was observed to be present in both the C. acuminata host and milestoe parasite, however, avonoids was signi cantly (P < 0.05) concentrated in the parasite as compared to the host plant (Table 2). Similarly, the parasite recorded considerably a signi cant (P < 0.05) amount of avonoids in P. macrophylla as compared to the host plant whereby avonoids was not detected. In contrast, avonoids was found in the host of C. sinensis but was not detected in the parasite ( Table 2). The total glycosides content recorded in the host and parasite of C. acuminata were not signi cantly different, while P. macrophylla and P. Americana had total glycosides content in their respective parasites but the different host had no glycosides (Table 3). Saponins was observed to be present in both the host and milestoe parasite of C. sinensis, however, it was signi cantly (P < 0.05) higher in the parasite as compared to the host plant (Table 4). Alternatively, saponins content was only found in the parasites of C. acuminata, P. macrophylla and P. Americana (Table 4).

Discussion
The aim of this study was to evaluate the phytochemicals present in the extracts of selected plants and T. globiferus at the host-parasite interface. The levels of the phytochemical contents evaluated varied signi cantly by plant species and their parasites. The in uence of the host plant on secondary metabolite levels of mistletoes is well-known. This has been associated to their parasitic habit that photosynthesizes but depends on the host for water and nutrients for photosynthesis. [14,15] It was observed that the parasite generally contained higher concentrations of the phytochemicals evaluated than their host species. This observation is similar to the report of Lo Gullo et al. [16] who reported higher concentrations of minerals in parasites than their host species. The variation in secondary metabolites had been described by Urban et al. [17] to be as a result of the transpiration rates of mistletoes being higher than that of the host plants. [9,15] However, the differences in phytochemicals in the wood of the host plant and the parasite at the interface could signify that they were two different plant species. More so, the differences in phytochemical contents in mistletoes from different host could also be in uenced by the environment the host plant faces, such as soil fertility. [15,18] Therefore, the inconsistent concentration of phytochemicals observed in our study could be due to the differences in species as well as in the environment.
Flavonoids defend plants against a variety of biotic and abiotic stresses by demonstrating a wide spectrum of biological functions and play a signi cant function in the interaction between the plant and their environment. [19] The increased avonoid content in C. acuminata and P. macrophylla could be an indication that the parasites were more exposed to environmental stress than the host. Studies show that avonoids could have a determinant role in response to different stresses such as drought. [20] In this regard, Nakabayashi et al. [21] reported that the over expression of some genes involved in avonoid metabolism and over accumulation of avonoids would caused an increase in drought tolerance. Therefore it would be reasonable to suggest that, the parasites utilizes this as a medium of survival since the transpiration rates of mistletoes are higher than that of the host plants. [17] Saponins are usually found in tissues that are highly susceptible to attacks by bacteria, fungi or insects. [22] Hence, it is assumed that one of the roles saponins plays in plants is to act as a chemical barrier in opposition to prospective pathogens. Therefore, this would account for their antimicrobial activity. [23,24] Due to their toxicity to various organisms, saponins can be utilised for their insecticidal, antibiotic, fungicidal, and pharmacological properties. [24] Conclusion At the host-parasite interface, signi cantly higher phytochemicals in the wood portion of Tapinanthus globiferus (milestoe) was observed as compared to the host plants wood, however, the variability in phytochemical content of Tapinanthus globiferus is dependent on the host. From these ndings, milestoe grown on C. acuminata and P. macrophylla will be rich in avonoids, while C. sinensis host favours the production of saponins in milestoe. Therefore, milestoe would be a better source of bioactive compounds with high medicinal values than their host plants if explored further.

Collection and Authentication of Plant Specimens
The twigs of mistletoes parasites from C. sinensis, P. macrophylla, C. acuminata and P. americana and their respective host stems were collected from farmlands in Alor-Agu, Igboeze South L.G.A, Enugu, Nigeria with the approval of the locals and authenticated and deposited by Mr. Felix Nwafor in the Department of Pharmacognosy and Environmental Medicines Habarium, University of Nigeria Nsukka, Enugu, Nigeria with voucher specimen number of PCG/UNN/00266 (Tapinanthus globiferus), PCG/UNN/0056 (C. sinensis), PCG/UNN/0080 (P. macrophylla), PCG/UNN/0330 (C. acuminata) and PCG/UNN/0012) P. americana. The plant specimens were cleaned, air-dried at room temperature and reduced to ne particles. The air-dried samples were pulverised and 10g of each samples were macerated with 100mL of methanol and kept at room temperature for 48hrs with agitation. The solvents were dried in vacuo at 40°C to obtain the dry extracts. [10] Qualitative phytochemical analysis of the pulverised samples was done to determine the presence of secondary metabolites ( avonoids, tannins, saponins, alkaloids, glycosides, terpenoids and steroids) according to standard procedure. [11] The con rmed metabolites where then further analyzed quantitatively.

Preparation of the Calibration Curves
Quercetin A quercetin stock solution was prepared by dissolving 10mg in 10mL methanol to give a stock solution of (1000 mg/L). The stock solution was further diluted to different concentrations of 100, 80, 60, 40 and 20mg/L. 1mL each of the different concentration of quercetin was mixed with 4mL of distilled water and 0.30mL of 5% (w/v) sodium nitrite. After 5 min, 0.30mL of 10% (w/v) AlCl 3 .6H 2 O solution was added to the mixture, followed by addition of 2mL of 1.0M NaOH after another 5min and diluted to 10mL with distilled water. The absorbance of the different concentrations was then measured against the reagent blank at 510nm with a UV/Visible spectrophotometer.
Digitoxin: The standard curve was prepared by adding 0.2mg/mL digitoxin in chloroform -methanol (1:1; v/v). A solution of 0.1, 0.2, 0.3, 0.4 and 0.5mL each (equivalent to 0.02, 0.04, 0.06, 0.08 and 0.10mg of digitoxin respectively) was transferred to a dry Erlenmeyer asks and evaporated to a small bulk (0.35mL) and made up to volume with distilled water. Freshly prepared Baljet's reagent (10mL) was added to each ask and allowed to stand for 1 h at room temperature and the resulting mixture diluted with 20mL of distilled water. The absorbance of the colour developed was determined using a spectrophotometer against a suitable blank at 495 nm.

Diosgenin
Diosgenin standard (0.20mg/mL) was dissolved in methanol and 0.1, 0.2, 0.3, 0.4 and 0.5mL (equivalent to 0.02, 0.04, 0.06, 0.08 and 0.10mg of diosgenin respectively) each of the standard solution was transferred to a dry test tubes. Vanillin-acetate solution (0.20mL of 5% (w/v)) was added, and followed by the addition of 0.80mL sodium perchlorate solution. The mixture was shaken and heated for 15mins in a water bath at 60 o C. After cooling, 5mL of glacial acetic acid was added to each test tubes and the absorbance of the mixtures determined against the blank using a UV/Visible spectrophotometer at 548 nm.

Determination of Total Flavonoids Content (TFC)
Aluminium-chloride colourimetric assay was used to determine the total avonoids content in the extracts as adopted by Agbo et al. [10] The extracts (1mg/mL) were prepared following the same procedure in preparing quercetin as reported above. The absorbance of the extracts was measured against the reagent blank at 510nm with a UV/Visible spectrophotometer. The total avonoids content was determined from the calibration curve and expressed as milligram of quercetin equivalent per gram of extracts (mgQE/g).

Determination of Total Glycosides Content (TGC)
Balet's reagent colourimetric method was used for the determination of the total glycosides content of the extracts as previously reported with slight modi cation by To ghi et al. [12] The extract (1mg/mL) was dissolved with 6mL of distilled water and 1mL of 12.5% (w/v) lead acetate solution was added. The mixture was made up to 10mL with distilled water and ltered. The ltrate (5mL) was transferred to a volumetric ask, 1mL of 4.77% (w/v) Na 2 HPO 4 solution added, and made up to 10mL with distilled water and ltered. Baljet's reagent (10mL) was added to 1mL of the clear ltrate and the mixture allowed to stand for 1h and diluted with 20mL of distilled water. The absorbance of the mixture was read against the blank at 495nm with a UV/Visible spectrophotometer. The total glycosides content was determined from the calibration curve and expressed as milligram of digitoxin equivalent per gram of extracts (mgDE/g).

Determination of Total Saponins Contents (TSC)
Vanillin-acetate colourimetric method was used for the determination of the total saponins content of the extracts as previously reported by Benyong et al. [13] with slight modi cation. A freshly prepared 5% (w/v) vanillin-acetate solution (0.20mL) was added to 0.10mL of the extract (1mg/mL) in a test tube followed by the addition of 0.80mL (w/v) sodium perchlorate solution. The mixture was shaken and heated for 15mins in a water bath at 60 o C and cooled. 5mL of glacial acetic acid was added to the mixture and the absorbance of the mixture read against the blank at 548nm using a UV/Visible spectrophotometer. The total avonoids content was determined from the calibration curve and expressed as milligram of diosgenin equivalent per gram of extracts (mgDE/g).

Data analysis
The entire quantitative tests were carried out in triplicates and data collected were for the host and parasite were subjected to Analysis of Variance (ANOVA) using IBM SPSS ver. 20.  Calibration Curve for Diosgenin Standards