Journal of Phytomedicine and Therapeutics www.niprdjopat.gov.net; niprdjopat@gmail.com Tarfa et al JOPAT Vol 21(1), 754 - 771, Jan – June. 2022 Edition ISSN2636 - 5448 https://dx.doi.org/10.4314/jopat.v21i1.3 Evaluation of safety of aqueous extract of Tapinanthus sessillifolius parasitic on Psidium guava Florence D.Tarfa, 2Onanuga C.O,3Obodozie-Ofoegbu. O . and 4Adoga G.I 1* Department of Medicinal Chemistry and Quality Control (MC & QC), National Institute for 1, 2,3 Pharmaceutical Research and Development (NIPRD), Idu Industrial Area, P.M.B 21 Garki, Abuja Nigeria Department of Biochemistry,College of Medicine,University of Jos,Nigeria. 4 Abstract Plants products have been a source of food and medicines since antiquity. Tapinanthus sessillifolius commonly known as African mistletoe is used in lifestyle preventive medicine to enhance wellbeing and for treatment of various diseases. The heavy metals, macro elements were determined using Flame Atomic Absorption Spectrophotometer and some physio-chemicals were also evaluated for consistency of the extract. The acute and subchronic toxicity studies of aqueous fresh leaf extract of Tapinanthus sessillifolius was evaluated in albino mice and rats. This is to determine its safety profile by evaluating its effects on feed and water intake, body weight, relative organ weight and changes in some biochemical parameters after 21 days daily oral administration to rats. The results, estimated LD50 to be greater than 2000 mg/kg/bw. The extract had no adverse effect on the efficiency of food and water consumption. Relative organ weight and the biochemical parameters tested were not significantly different p<0.05 when compared to untreated animals. This was supported by histopathological studies of the organs where no adverse lesions were observed on tissues. However, there were lymphatic aggregates infiltration in one of the lungs rat treated with 800mg/kg. The toxic heavy metals, lead, cadmium and arsenic were not detected while moisture and ash were 7.02 % and 10.2% respectively falling within the permissible limit of WHO and RDA. Repeated oral administration of fresh leaf extract of Tapinanthus sessillifolius is relatively safe. Keywords: Tapinanthus sessillifolius, Psidium guajava, safety, heavy metals, biochemicals. ©2007 The authors. This work is licensed under the Creative Attribution 4.0 International license. 754 Journal of Phytomedicine and Therapeutics Tarfa et al www.niprdjopat.gov.net; niprdjopat@gmail.com Introduction Infestation of mistletoes on smaller trees The use of medicinal plants as remedies for lead to poor growth and productivity and treatment of diseases, fitness, wellness and eventual death of host plants, especially longevity are becoming more popular with during unfavorable weather conditions. the emergence of COVID -19 pandemic and Ethnomedicinal uses of mistletoes had for a other chronic ailments. Mistletoe with the very long time been in the hands of very few common name(s) – bird lime, all heal, herbal practitioners who claimed general use devil’s fuge, Iscador is an all heal plant to sorcery and magical powers to treat [1,2,3]. Mistletoe is a general term for mental conditions, sterility, and health woody shoot parasites in several plant families, especially Loranthaceae problems and belong to the ones majorly used in herbal medicine for the treatment of headache, rheumatic pain, Loranthaceae are Helixanthera, Berhautia, Globimetula, urino-genital leaves, stems, berries and flowers are the family Loranthaceae (5,6). The seven genera of the Englerina, with system, rheumatism and pain. [4,8]. The Viscaceae [4,5]. Most genera of African mistletoes associated hypertension [10,11] ulcers [12] and cancers Agelanthus, [13,14]. They are also acclaimed to possess Tapinanthus and Phragmanthera with about hypoglycemic [15,16] lipid lowering [17] five or more species are recognised in West and antibacterial effects [18]. Africa [7]. Worldwide, approximately 1500 species of mistletoe have been identified The chemical information readily available [7,8.]. Mistletoes are hemi-parasitic shrubs includes the structural polysaccharide and growing on dicotyledonous trees such as protein in Phragmanthera capitata leaf cell Albizzia leb beck, Terminalia mantaly, wall, the viscotoxins, carbohydrates and Terminalia catappa, Khaya senegalensis, cytotoxic lectins. Compounds like alkaloids, and flavonoids, cyanogenic glycoside (linmarin Theobroma cacao [7]. Mistletoe attaches gallate) and walbruside were isolated [19]. itself to the host by modified roots otherwise Flavonoids namely, quercetin, catechin, known as “haustorium” [5,6,7,]. Haustorium qercitrin, rutin and avicularin have been ensures the continuity of the macro elements isolated from the leaves of Globimimetula sodium (Na), potassium (K), calcium (Ca) braunii [19,20]. The present study is to from the host to the parasite plant [8,9]. evaluate the acute and subchronic toxicity Citrus grandis, Cola acuminata 755 Journal of Phytomedicine and Therapeutics Tarfa et al www.niprdjopat.gov.net; niprdjopat@gmail.com effects of the aqueous fresh leave extract of Moisture - The determination of on moisture content was based on the Psidium guajava host and also determine method of the Association of Official some quality standard makers of ash, Analytical Chemist (AOAC) [20]. It moisture and toxic heavy metals. is an indirect distillation method Tapinanthus growing A. sessilifoliuss (evaporation of moisture). One gram Materials and methods (1.0 g) of the sample powder was Plant material weighed on aluminum foil on the The leaves of T. sessilifolius were harvested automated moisture analysis pan from the host, Psidium guajava in Jos, (Model MB 200, OHAUS Florham Plateau State of Nigeria. The plant material PK.USA) and set at 105 °C for 3 was identified and authenticated by Prof. Z. hours O. Gbile (Consultant Taxonomist, UNDP). moisture content of the sample was Voucher specimen (No. FHI 105336) was obtained from the moisture balance deposited at Forestry Research Institute, B. Ibadan and at NIPRD, Idu, Abuja Nigeria. after which percentage Total Ash - The total ash was also determined by AOAC [20]. Porcelain crucible was washed and Preparation of extract Extraction of placed in the muffle furnace for 10 Plant minutes to dry. After which it was Tapinanthus sessillifolius removed and placed in a desiccator The fresh plant material was cold extracted containing active desiccant to avoid with distilled by disruption of the cell wall moisture contact and allowed to cool. using blender. The extract was filtered After cooling, it was weighed (M1). 2 through muslin cloth then filtered again g of each of the sample were through and weighed in the crucibles (M2) and lyophilized using Lyovac GT2 (Germany). was placed in the Muffle furnace at a The yield was found to be (10.53 % w/w). 550 oC and allowed to stand 3 hours Whatman filter No. 42 for complete combustion to ash to be Physical Evaluation achieved. The ash samples were Proximate analysis of extract removed placed in the desiccator for cooling and the weighed (M3). The 756 Journal of Phytomedicine and Therapeutics Tarfa et al www.niprdjopat.gov.net; niprdjopat@gmail.com percentage ash was calculated using Atomic Absorption Spectrophotometer the formula: (AAS) using air acetylene as fuel. The slit of 0.5 and appropriate lamps and their wavelengths were used. The mean value and the standard deviation of each element C. was determined by the AAS computer. Total proteins were estimated by the Samples were analyzed in triplicate. Results method of AOAC [20] obtained in part per million (PPM) were converted Phytochemical screening preliminary phytochemical Animals flavonoids, alkaloids and terpenes [21,22]. Swiss albino mice (20 – 30 g) and Wistar Elemental analysis rats (150 – 200 g) of either sex obtained and Plant samples were prepared for elemental maintained at the Animal Facility Centre analysis as modified by AOAC [20]. The (AFC) dried plant extract was ashed in oven of the National Institute for Pharmaceutical Research and Development, electric muffle furnace maintained at 400 C o Abuja were used. All animals were housed and 420 oC, for about 6 - 7 h to destroy all under standard conditions of temperature organic materials in the sample. The (25 ± 2 oC) and light approximately (12/12 h crucible containing pure ash was then taken light/dark cycle) and fed on standard diet out of the furnace and kept in a desiccator. and water ad libitum. These animals were Thereafter the ash was digested with triple acids: as In-vivo toxicity studies screening for phenols, tannins, saponis, mixture gram were determined by flame flourimeter. and Agrawal and Paradhavi (2007) were for milligram/100 indicated below. Sodium and potassium The standard methods of Harbone (1998) used to approved for use by the ACE committee sulfuric after reviewing the protocol for good acids:sulphuric:perchloric acid (11:6:3) to laboratory practice and animal handling, obtain a clear solution. The solution was which is in compliance with the National then made to 25 ml with double distilled Institutes of Health Guide for the Care and water and read up with the flame technique use for Laboratory animals (Publication No. of Hitachi Model 80-80 polarize Zeeman 85-23, revised 1985(23). 757 Journal of Phytomedicine and Therapeutics Tarfa et al www.niprdjopat.gov.net; niprdjopat@gmail.com ��� = Acute toxicity tests The oral acute toxicity (LD50) ����ℎ������������������ℎ������ �100 ���������ℎ����ℎ������ ��� = was determined in mice by a modified Lorke’s ������������������������ℎ������ �100 ���������ℎ����ℎ������ Animals [24] model. The test was carried out in five were observed for signs of abnormalities during the whole treatment. groups, each consisting of five (5) mice. Animals were fasted overnight after of food Group I – IV were administered with the last dose, but allowed free access to varying doses of extract at 10, 100, 1000 and water. Blood samples were obtained by 2000 mg/kg i.p., while group V served as retro-orbital puncture, using capillary tubes control and was administered normal saline for blood biochemical studies after which at 10 ml/kg. Signs and symptoms of toxicity the animals were anesthetized by chloroform was observed over a period of 24 h. Death inhalation. The internal organs were isolated, and sign of toxicity within this period were weighed and fixed in formaldehyde for monitored was recorded. histopathological investigation. The relative organ weight (ROW) was calculated thus: Sub-Chronic toxicity Studies Test Guidelines with slight modifications ��� = according to the Organization for Economic Cooperation and Development (OECD, 407) [25]. Healthy rats of either sex were ������������ℎ��������(�) �100 ��������ℎ����������������������(�) Statistical analysis: Data were expressed as randomly divided into four groups (n= 5). Mean ± SEM Data for toxicity studies were Animals received vehicle orally (water; analyzed by ANOVA followed by Dunnett’s control group) and aqueous extract in doses post hoc test for multiple comparisons and of 200, 400 and 800 mg/kg/day for 21 p<0.05 was considered significant. consecutive days. Body weight was recorded weekly as D1, D7, D14, D21. Food Results consumption and water intake were daily Phytochemical monitored. Daily feed (DFI) and Daily water analysis intake (DWI) were determined [30] as: 758 and Physicochemical Journal of Phytomedicine and Therapeutics Tarfa et al www.niprdjopat.gov.net; niprdjopat@gmail.com The extract of protein and fats that were present are shown the in Table 2. Table 3 represent the mineral polyphenols, terpenoids saponin, glycosides. elements and heavy metals that were not Alkaloids were not detected as shown in detected. secondary Table 1. showed the metabolites Moisture, Table 1. presence such ash, as carbohydrate, Phytochemical compounds of aqueous fresh leaf extract of Tapinanthus sessilifolius on P. guajava host plant Phytochemicals Remarks Polyphenols Flavonoids ++ ++ Terpenoids Saponins ++ Alkaloids ++ - ++ = Positive; - = Not detected Table 2. Some physico-chemical parameters of aqueous fresh leaf extract of Tapinanthus sessillifolius on P.guajava host plant Parameter Ash Moisture Lipids Protein Carbohydrate Fibre Concentration (%) 10.02 7.02 16.32 5.56 54.8 7.3 WHO <8 <10 - - - - 759 Journal of Phytomedicine and Therapeutics Tarfa et al www.niprdjopat.gov.net; niprdjopat@gmail.com Table 3: Mineral and heavy metal content of aqueous fresh leaf extract of Tapinanthus sessillifolius Elements Na (mg/100g 1.22 ± dry 0.01 weight) RDA mg WHO <2300 K 32.1 ± 0.2 Mg Ca 10.95 ± 50.55 ± 0.01 0.42 Fe Zn Pb Cd As 7.89 ± 0.02 13.42 ± 0.009 ± 0.02 0.01 ND ND 0.4-5.1 30-40 2001300 0.2-27 2-15 - - - - - - 50 10 - - - RDA means recommended daily allowance (DRI, Food and Nutrition Board, 2000) WHO = World Health Organization ND = Not detected Effect of leaf extract T. sessillifolius on Effects on sub-chronic toxicity studies in acute oral administration in mice rats The oral acute administration of the extract The at varying doses of 10, 100,1000, and 2000 sessillifolius at doses of 200, 400 and 800 mg/kg showed no any signs of toxicity. The mg/kg bw. for 21 days did not show behavioural pattern was similar in treated significant difference in body weight when groups and control animals. No death was treated animals were compared to normal recorded in all animals. The acute lethal control group even though there is increase dose (LD50) was therefore estimated to be in weight across all groups (Table 4). Also, greater than 2000 mg/kg body weight. there was no significant change in food and 760 daily oral administration of T. Journal of Phytomedicine and Therapeutics Tarfa et al www.niprdjopat.gov.net; niprdjopat@gmail.com water intake with administration of the Microscopic examination of the tissues of extract. The relative organ weights of liver, the internal organs did not show any change kidney, lungs, spleen and pancreas treated in the structure of the cells of liver, kidney rats were similar to control with no spleen and pancreas of the treated animals significant difference between the groups that could be ascribed to the treatment of (Table 5). Treatment with the extract did not rats with the aqueous leaf extract of T. affect the levels of serum glucose or lipid sessillifolius. profile as all the parameters testes were aggregates were observed in one of the lung within normal range and not significantly tissue in treated animals at doses of 800 different from untreated group (Table 6). mg/kg which were not present in control However, lymphocytic animals (Figure 1). Histological analysis Table 4. Effect of aqueous leaf extract of Tapinanthus sessilifolius (TS) on relative organ weight (g) in normal rats Organ (g) TS (mg/kg) Liver Kidney Lungs Spleen Pancreas Heart Control 3.30 ± 0.10 0.96 ± 0.11 0.75 ± 0.14 0.37 ± 0.30 0.153 ± 0.20 0.35 ± 0.21 200 3.16 ± 0.20 0.91 ± 0.12 0.69 ± 0.13 0.34 ± 0.11 0.16 ± 0.09 0.32 ± 0.06 400 3.21 ± 0.11 0.89 ± 0.20 0.72 ± 0.21 0.33 ± 0.02 0.151 ± 0.06 0.33 ± 0.12 800 3.32 ± 0.11 0.94 ± 0.10 0.77 ± 0.12 0.32 ± 0.11 0.153 ± 0.2 0.33 ± 0.12 Data are expressed as Mean ± SEM (n = 5). All Values in test group are not significantly different from control (p<0.05) Two-way ANOVA Dunnett post hoc. 761 Journal of Phytomedicine and Therapeutics Tarfa et al www.niprdjopat.gov.net; niprdjopat@gmail.com Table 5: Effect of Aqueous leaf extract of Tapinanthus sessilifolius (TS) on glucose and lipid profile in normal rats. Lipid profile (mg/dl) TS (mg/kg) Glucose Cholesterol HDL-cho LDL-cho Triacylglycerol Control 94.34 ± 2.1 97.00 ± 1.5 39.10 ± 1.3 39.70 ± 0.9 109.50 ± 1.3 200 95.82 ± 5.2 92.80 ± 5.2 38.90 ± 1.7 39.10 ± 1.5 102.50 ± 1.5 400 93.25 ± 2.2 89.80 ± 4.2 40.20 ± 0.8 39.10 ± 1.2 95.50 ± 1.3 800 98.33 ± 3.1 91.34 ± 1.7 39.1 ± 2.0 39.70 ± 1.5 107.03 ± 1.2 Data are expressed as Mean ±SEM (n = 5). All values in test group are not significantly different from untreated control (p<0.05). Two-way ANOVA Dunnett post hoc. 762 Journal of Phytomedicine and Therapeutics Tarfa et al www.niprdjopat.gov.net; niprdjopat@gmail.com Table 6. Effect of aqueous leaf extract of Tapinanthus sessilifolius (TS) on some biochemical parameters in normal rats. TS (mg/kg) ALT(UI) AST(UI) Ratio Creatine (UI) ALP T.BIL Control 20.31± 0.70 39.6 ± 0.1 0.33 0.37 ± 0.30 0.153 ± 0.20 2.40 ± 0.31 200 19.5 ± 0.10 38.5± 0.12 0.45 0.34 ± 0.12 0.160 ± 0.09 2.32 ± 0.41 400 19.80 ±1.6 39.50± 0.2 0.51 0.33 ± 0.2 0.154 ± 0.11 3.12 ± 0.21 22.4 ± 0.5 40.20 ±0.1 0.55 0.32 ± 0.1 0.151 ± 0.1 3.20 ± 0.4 800 Data are expressed as Mean ± SEM (n = 5). All values in test group are not significantly different from untreated control (p<0.05). Two-way ANOVA Dunnett post hoc. 763 Journal of Phytomedicine and Therapeutics Tarfa et al www.niprdjopat.gov.net; niprdjopat@gmail.com Control TS (800 mg/kg) A - Normal Lung Figure 1 B - Focal lyphocytic aggregate Representative slides of the histological analysis of the lung tissues of control (A) and treated (B) animals (Mag 20x10) Discussion Plants are a rich source of macro elements essential for human beings and they Toxicological studies are generally done to contribute to vital body function growth [29]. determine the level of exposure and prevent In this study, the inorganic essentials the potential risk associated with the use of elements such as sodium (Na), potassium medicinal plants. The low level of moisture (K), calcium (Ca) and the trace elements that content that falls within the acceptable limit include iron (Fe), magnesium (Mg) and zinc of 10 % will not promote microbial growth and subsequent biodegradation (Zn) and which are required for various metabolic processes in man were detected instability of the bioactive components of and the values falls within the WHO limits the plants [27,28]. Fibre content of 7.3% is [43]. Adding to the integrity and safety of good for proper motility and faecal excretion. the extract is the absence of heavy metals 764 Journal of Phytomedicine and Therapeutics Tarfa et al www.niprdjopat.gov.net; niprdjopat@gmail.com like Lead (Pb), Cadmium (Cd) and Arsenic nutritional composition might have played a (As). These metallic elements were not significant detected in this plant material as reported for degenerative diseases and processes [34]. other herbals [29,30]. Decrease in weight or excessive increase can According to reports by Rosidah 2009 [31] be a pointer to toxicant in the system. administration of 500 - 5000, and 5000 - However, the treatment with fresh extract 15000 mg/kg body weight is relatively safe the weight gain is not different from and non-toxic respectively. Therefore, the untreated even though not with food and lethal dose of greater than 2000 mg/kg of water efficiency and relative organ weight. this study is relatively safe. The repeated The astringency and pungency of the fresh administration of doses up to 800 mg/kg to extract would have caused the moderate normal rats did not produce observable toxic consumption especially at higher dose of effects at the doses tested. This is consistent 800mg/kg body weight and the possibility of with other reports which showed that the mild lymphatic aggregates infiltration mistletoes extracts are relatively non-toxic observed in the lungs histopathology of one [30,31,32]. However, in handling the plant in this treated group and this may be during material from raw material to extract, oral administration liquid entered the lung. physicochemical reference like moisture and The liver and kidney play vital role in ash are critical so as to avoid deterioration, biotransformation and filtering blood from microbial and fungal growth that can be digestive tract to rest of the body [36] introduced as toxicants [33]. The extract is Therefore, rich in polyphenols that are strong radical inflammation scavengers or antioxidants [34] but lack mitochondrial cells. It is known that alkaloids which was reported by other elevated studies on same host and difference species conjunction with a rise in bilirubin level are and this could be due to season and location considered of harvest [30;32] This is in agreement to hepatotoxicity linked to oxidant stress [35]. the chemotaxanomic profiles [41]. Increase in the aminotransferases in serum The quality of many foods depends on the indicates cellular injury that leads to concentration and type of minerals and leaching of enzymes to serum before its biomolecules they contain. The extracts manifestation with clinical histopathology 765 role any against toxicants and a can injury transaminase as variety of cause to activities marker index the in of Journal of Phytomedicine and Therapeutics Tarfa et al www.niprdjopat.gov.net; niprdjopat@gmail.com [36]. Reduced clearance due to increased The work was supported by NIPRD proliferation of cells, increased rate of cell postgraduate turn over and increased cell damage and thankful to Mr Sunday Dzarma for technical increase in enzyme synthesis [37,38] This assistance , Mrs Faustina Nwachikwu , Mr increase was not the case in this studies Bamaiyi Wayas (of blessed memory) and since the enzyme ratios were less than one Ali Sale of the Animal Facility Centre of because ratio of one indicates injuries and Department damage to liver [39]. It has been frequently Toxicology, NIPRD. reported that in the development of liver oxygen species Pharmacology are and often 1. Abayomi, S., Eyitope, O. and Adedeji, leading to greater hepatic lipid peroxidation. O. (2013). The role of medicinal plants Lipotropic in mediators (ROS), of Authors References diseases there is an increased production of reactive sponsorship. and intracellular strategies for disease signals activate Kupffer cells, which initiate prevention.Tradit .Complement Altern and perpetuate the inflammatory response Med. and development of fibrosis and leaching of http://dx.doi.org/10.4314/ajtcam.v10 enzymes out to blood. [39,40,41]. The i5.2 extract can be said to at no adverse effect 2. Craig, level from the observed reports. 10(5): W.J. 210-229. (2001). Health Promoting Herbs as Useful Adjunct Conclusion to Prevent Chronic Diseases. In: The administration of the aqueous fresh Disease Prevention. Wilson, T. and leave extract of mistletoe T.sessililfolius N.J. Temple Edn. Humana Press Inc., parasitic on Psidium guava is relatively safe New Jersey, 237. Nutritional Health: Strategy for within the limit of this studies. This validates its folkloric use of the plant for 3. 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