Effect of Aqueous Extract of Khaya Grandifoliola C. DC. Stem Bark on Some Disaccharidases Activity in Iron-Deficient Weanling Rats

Asian Journal of Research in Biochemistry

7(3): 1-7, 2020; Article no.AJRB.60874 ISSN: 2582-0516

Effect of Aqueous Extract of Khaya grandifoliola C. DC. Stem Bark on some Disaccharidases Activity in Iron-Deficient Weanling Rats

Emmauel Gboyega Ajiboye1* and Temidayo Adenike Oladiji1

1Department of Biochemistry, University of Ilorin, Ilorin, Nigeria.

Authors’ contributions

This work was carried out in collaboration between both authors. Author EGA designed the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the manuscript. Authors EGA and TAO managed the analyses of the study. Author EGA managed the literature searches. Both authors read and approved the final manuscript.

Article Information

DOI: 10.9734/AJRB/2020/v7i330138 Editor(s): (1) Dr. Mohamed Fawzy Ramadan Hassanien, Zagazig University, Egypt. Reviewers: (1) Eugenia Henriquez-D’Aquino, University of Chile, Chile. (2) B. Kumudhaveni, Madras Medical College, India. Complete Peer review History: http://www.sdiarticle4.com/review-history/60874

Received 27 June 2020 Original Research Article Accepted 03 September 2020 Published 08 September 2020

ABSTRACT

This study was aimed at investigating the effect of the aqueous extracts of Khaya grandifoliola stem bark on some disaccharidases (lactase and sucrase) in diet-induced anemia in weanling rats. Weanling rats of 21 days old were maintained on iron-deficient diets for four weeks to induce anemia before treatment. A total of 35 weanling rats were used, grouped into five rats per group of iron-deficient diet/distilled water, iron-sufficient diet/distilled water, change of iron-deficient diet after four weeks to iron-sufficient diet, iron-deficient diet/ Standard drug, iron-deficient diet/25 mg/kg body weight of aqueous plant extract, iron-deficient diet/50 mg/kg body weight of aqueous plant extract, and iron-deficient diet/100 mg/kg body weight of aqueous plant extract. Phytochemical screening of the extract revealed the presence of alkaloids, saponins, cardiac glycosides, tannins, anthraquinones and flavonoids. The extract administered orally produced significant increase in the activity of intestinal mucosa sucrase and lactase (P < .05). The change of diet from iron deficient diet to iron sufficient diet increased disaccharidases activities in the intestinal mucosa. However, the aqueous extract of Khaya grandifoliola showed a higher disaccharidases activity when compared to the group of rats that were fed iron-sufficient diet. This study revealed that plant extract administered ______

*Corresponding author: E-mail: [email protected];

Ajiboye and Oladiji; AJRB, 7(3): 1-7, 2020; Article no.AJRB.60874

increased disaccharidases activity in the intestinal mucosa in diet-induced anemic group of weanling rats and thus lends credence to Khaya grandifoliola use in folklore medicine in the management of anemia.

Keywords: Khaya grandifoliola; iron-deficient; lactase; sucrase.

1. INTRODUCTION 2. MATERIALS AND METHODS

Iron deficiency anemia (IDA) is the most common 2.1 Experimental Animals type of anemia that results from an inadequate iron supply to aid the production of healthy red Forty-five weanling albino rats of both sexes blood cells [1]. Iron deficiency is usually a result (Rattus norvegicus) with a mean weight of 40.0 g of the depletion of the body's iron stores; hence, ±3.0 g were obtained from the Animal Holding a restricted supply of iron to various tissues Unit of the Department of Biochemistry, becomes apparent. This may result in a University of Ilorin, Nigeria. reduction in the synthesis of red blood cells and iron-dependent intra-cellular enzymes 2.2 Feed Components participating in many metabolic pathways [2].

Iron deficiency has been reported to reduce the Yellow maize (Zea mays) and locust bean activity of some disaccharidases [3,4]. (Parkia biglobosa A. Jacq) seeds were obtained Although there are various drugs used for the locally from Baboko Market, Ilorin, Nigeria, while treatment of anemia [5,6,7], they are not the soybean oil used was a product of Grand affordable to many poor people in the developing Cereals and Oil Mills Limited, Bukuru, Jos, countries such as Nigeria. Furthermore, the rural Nigeria. The vitamin mix was a product of BASF populations in various parts of the world do not Aktiengesellschaft, Germany Pantex, Netherland. have appreciable access to high-quality drugs for Component chemicals of the mineral mix used the treatment of anemia, hence, the reliance on were products of Sigma-Aldrich Chemical herbal products for the treatment of diseases and Limited, London, UK. anemia. Some of the plants with ethnobotanical and scientific claims being used for the treatment 2.3 Reagents of anemia include Waltheria indica, Sorghum bicolor, Khaya grandifoliola, and Mangifera indica The reagents used were of analytical grade and [8,9,10,11,12]. were prepared in all glass-distilled water. The reagents were stored in reagent bottles [17]. Khaya grandifoliola is a medicinal plant commonly found in Nigeria and many parts of the 2.4 Plant Identification and Preparation of plant are valuable to traditional medicine. Some Extract sicknesses like gastric pain, lumbago, stomach ache, worm infestation, cough, fever, and The stem barks of the plant were obtained from rheumatism are treated with concoctions made Ikaro in the Ose Local Government Area of Ondo from the bark of Khaya grandifoliola [13]. The State in the South West region of Nigeria. It was stem bark of this plant has also been previously authenticated in the Department of Plant Biology, reported to have anti-malarial, anti-ulcer, anti- University of Ilorin, Ilorin, Nigeria, where a hypoglycaemic, anti-hypoproteinaemia, and anti- voucher specimen was deposited in the hypocholesterolaemia activity [14,15,16]. Several herbarium with voucher number UILH/003/1066. research studies have lent scientific credence to the use of various parts of Khaya grandifoliola in The method described by Oladiji et al. [9] was the treatment of several diseases, however, used in the preparation of the plant extract. The there is still no adequate information on the effect stem barks of the plant were air-dried until a of the plant extract on lactase and sucrase constant weight was obtained. The dried pieces activity as an indicator for anti-anemic potentials were then pulverized using an electric blender, of Khaya grandifoliola. This study investigated and the powder obtained was stocked in a plastic the effect of aqueous extract of Khaya container. A known weight (15 g) of the powder grandifoliola stem bark on lactase and sucrase was poured into 100 ml of distilled water and activity in the intestinal mucosa of iron-deficient immediately boiled for 25 min. The resulting weanling rats. solution was left to cool and filtered with

Ajiboye and Oladiji; AJRB, 7(3): 1-7, 2020; Article no.AJRB.60874

Whatman filter paper. The filtrate was B – 35 weanling rats maintained on an iron- concentrated on a water bath (kept at 45°C) until deficient diet designated as IDF. a constant weight was obtained to give 2.32 g ± 0.03 of the residue (brownish-black slurry), which Animals in groups A and B were maintained on corresponded to percentage yield of 15.47%. their respective diets for four weeks. At the end The residues were pooled together and of the four weeks feeding period, five rats each reconstituted in distilled water to give the doses from ISF and IDF groups were sacrificed. The of 25, 50, and 100 mg/kg body weight. remaining weanling rats in groups B were further grouped into six with five rats in each group as The reconstituted aqueous extract was follows: administered orally using an oropharyngeal cannula to all the animals in different groups. B1- Iron deficient weanling rats fed with iron- deficient feed for two weeks (iron deficient 2.5 Diet Formulation feed all through) designated as IDF

The composition of iron-deficient and iron- B2- Iron deficient weanling rats fed with iron sufficient diets per kg diet is described in Table 1. sufficient feed for two weeks (change of The components of the diets were thoroughly feed) designated as COF mixed and made into pellets to ensure homogeneity and proper handling by the B3- Iron deficient weanling rats orally animals. It was produced weekly and packed into administered daily for two weeks with iron air-tight polythene bags to prevent rancidity, supplement syrup (a standard iron auto-oxidation of the oil, and microbial supplement) designated as SD contamination. The proximate analysis of the compounded feeds was also carried out. B4- Iron deficient weanling rats orally administered daily for two weeks with 25 2.6 Animal Grouping and Extract mg/kg body weight of aqueous extract of Administration Khaya grandifoliola stem bark designated as KG 25 mg. The animals were kept in well-ventilated house conditions (temperature: 22±3°C; photoperiod: B5- Iron deficient weanling rats orally 12 h/12 h light/dark cycle; relative humidity: 45- administered daily for two weeks with 50 50 %). They were allowed free access to normal mg/kg body weight of aqueous extract of rat chow and distilled. The acclimatization was Khaya grandifoliola stem bark designated as done for seven days, and they were then fasted KG 50 mg. for 24 hours (without food but water) before the B6- Iron deficient rats orally administered commencement of the experiment. The animal daily for two weeks with 100 mg/kg body grouping consisted of an initial two groups: weight of aqueous extract of Khaya A – 10 weanling rats maintained on iron- grandifoliola stem bark designated as KG sufficient diet designated as ISF. 100 mg.

Table 1. Feed components of iron sufficient and iron-deficient diets

Feed components Iron-sufficient (g/kg) Iron-deficient (g/kg) Locust bean seed 500 500 Maize flour 315 315 Soybean oil 40 40 Sucrose 100 100 Methionine 5 5 Mineral mixb 30 30 Vitamin mixa 10 10 FeSO4·7H2O 157.36mg/Kg 38.216 mg/Kg Soybean oil: polyunsaturated fatty acids (58%), monounsaturated fatty acids (29%), saturated fatty acids (13%). aVitamin mix (per kg of diet): vitamin A, 100,000 lU; vitamin D3, 10,000 lU; vitamin E, 100 mg; vitamin B1, 20 mg; vitamin B2, 40 mg; Lysine, 10g; d-calcium pantothenate, 100 mg; vitamin B6, 15 mg; vitamin C, 250 mg; vitamin K3, 15 mg; folic acid, 5000 mcg; nicotinic acid, 200 mg; biotin, 150 mcg; inositol, 80 mg. b Mineral mix (g/kg): CoCl2·6H2O (0.001), CuSO4·5H2O (0.079), MnSO4·7H2O (0.178), KI (0.032), NaCl (3.573), ZnCO3 (1.60), CaSO4 (11.610), MgSO4·7H2O (2.292), K2HPO4 (10.559). Control diet contained 1.078 g FeSO4·7H2O

Ajiboye and Oladiji; AJRB, 7(3): 1-7, 2020; Article no.AJRB.60874

The rest of the animals in group A were 3.1 Effect of Khaya grandifoliola on still fed with iron sufficient feed for two Intestinal Mucosa Sucrase Activity in weeks (iron sufficient all through) designated as Iron-Deficient Weanling Rats ISF. From Table 3, there was significant decrease in The extracts and the distilled water were the intestinal mucosa sucrase activity in IDF administered to the various groups using the oro- group when compared to that of ISF group. The pharyngeal cannula. activity of sucrase in the SD group and ISF group showed a significant increase when compared 2.7 Determination of Lactase and Sucrase IDF group. Also, there was also a significant Activity increase in sucrase activity in COF and SD groups when compared to IDF group. The result Method described by Dahlqvist [18] was used also showed a significant increase in the activity and the result of the intestinal mucosa of sucrase in COF group when compared with disaccharidases was reported as units (U). both SD and ISF groups. Administration of the aqueous extracts of K. grandifoliola stem bark led to significant (P<.05) increase in the activity 2.8 Statistical Analysis of the intestinal mucosa Sucrase (Table 3) at all dose levels investigated compared to the other Results were expressed as the mean ± SEM of groups. However, the dosage at 50mg/kg body five determinations. The data were analyzed weight of aqueous extract of K. grandifoliola using Duncan Multiple Range Test and stems bark has the highest significant increase complemented with Student's t-test. The (P<.05) in the activity of intestinal mucosa differences were considered statistically Sucrase. significant at P<.05. All the analyses were done using SPSS version 16.0 software (SPSS Inc., Table 3. Effect of Khaya grandifoliola on Chicago, IL, USA). intestinal mucosa sucrase in iron deficient weanling rats 3. RESULTS Group Sucrase specific Phytochemical screening of the aqueous stem activity (U/g ) a,b barks of K. grandifoliola revealed the presence of ISF 9.00±1.7 a alkaloids, flavonoids, tannins and saponins, IDF 5.27±0.2 a,b cardiac glycosides, and anthraquinones in its COF 9.13±1.4 a,b aqueous extracts. SD 6.93±0.3 a,b 25mg/kg b. wt. 8.93±0.2 c The proximate composition of both iron-deficient 50mg/kg b. wt. 16.20±1.6 b (ID) and iron sufficient (IS) formulated feeds are 100mg/kg b. wt. 9.67±1.7 shown in Table 2. Proximate analysis of the diets Values are expressed as mean of five replicates ± showed that the components in the iron- S.E.M. and those with different superscripts along a column are statistically different (P<.05). The extract deficient formulated feed (ID) were substantially was administered for 14days; IDF: Iron-deficient diet similar to those in the iron-sufficient formulated fed rats; ISF: Iron-sufficient diet fed rats; COF: Change feed. of feed to iron-sufficient diet; SD: Group treated with Standard Drug; Kg b. wt.: Kilogramme Body weight Table 2. Proximate composition of iron- deficient and iron-sufficient diets 3.2 Effect of Khaya grandifoliola on Intestinal Mucosa Lactase in Iron Components ID (%) IS (%) Deficient Weanling Rats Crude protein 20.78 21.44 Carbohydrate 60.00 59.65 From Table 4, lactase activity showed a Lipid 5.03 5.15 significant decrease in IDF group when Crude fiber 2.97 3.02 compared with ISF group. SD group has Total ash 4.20 4.35 significant increase in the intestinal mucosa Moisture 7.02 6.39 lactase activity when compared with both ISF ID, iron-deficient; IS, iron-sufficient; crude protein was and COF groups. In this study, the SD group obtained using the expression nitrogen×6.25 showed a significant increase in lactase activity

Ajiboye and Oladiji; AJRB, 7(3): 1-7, 2020; Article no.AJRB.60874

when compared with ISF group. This result also deficient diet in the study. Similar reports have showed a significant increase in lactase activity shown that dietary iron deficiency can cause a in COF group when compared IDF group. significant decrease in disaccharidase activity Administration of the aqueous extract of K. [3,19,20,21,22,23,24]. grandifoliola stem bark led to significant increase in the activity of intestinal mucosa lactase (Table Iron-deficient diet fed rats treated with the 4) at all doses investigated compared to other reference drug and the group of iron deficient groups (P<.05). However, the dosage at weanling rats administered aqueous extract of K. 50mg/kg body weight of aqueous extract of K. grandifoliola stem bark showed significant grandifoliola stems bark has the highest increase in the activity of disaccharidases in the significant increase in the activity of intestinal intestinal mucosa. This result is in agreement mucosa lactase (P<.05). with claim that, decrease in disaccharidases activity is reversible by iron supplementation Table 4. Effect of Khaya grandifoliola on [3,21,24]. intestinal mucosa lactase in Iron-deficient weanling rats The change of diet from iron deficient diet to iron sufficient was also able to increase the activities of intestinal mucosa sucrase and lactase, Group Lactase specific however, the plant aqueous extract of K. activity(U/g) a,b grandifoliola showed higher increase in the ISF 4.13±1.0 activity of the disaccharidases. IDF 1.73±0.9a a,b COF 3.27±2.0 The significant increase in the activities of the SD 5.73±1.8a,b a,b disaccharidases in the iron-deficient weanling 25mg/kg b. wt. 4.93±0.7 K. a,b rats from administered aqueous extract of 50mg/kg b. wt. 7.13±1.3 grandifoliola stem bark in this study may be as a a,b 100mg/kg b. wt. 5.87±0.8 result of the chemical constituent(s) of the of the Values are expressed as mean of four replicates ± aqueous extract of K. grandifoliola stem bark. It S.E.M. and those with different superscripts along a is possible that the extracts mechanism(s) of column are statistically different (P<.05). The extract action for the significant increase in the activities was administered for 14days; IDF: Iron-deficient diet fed rats; ISF: Iron-sufficient diet fed rats; COF: Change of the disaccharidases may be due to an of feed to iron-sufficient diet; SD: Group treated with increase in the gene expression of both sucrase Standard Drug; Kg b.wt.: Kilogramme Body weight and lactase. Since the extract contains saponins and they are membrane active agents. It is 4. DISCUSSION possible that saponins stimulate the release of glucocorticoids that further initiate various Secondary metabolites screening carried out on biochemical reactions, hence, resulting in the the aqueous stem barks of K. grandifoliola induction of the disaccharidases in the intestinal indicated that, K. grandifoliola stem barks contain cells. alkaloids, flavonoids, tannins and saponins, and The administered doses of aqueous extract of anthraquinones in its aqueous extracts [13]. Khaya grandifoliola in this study were within the These phytochemicals are known to perform tolerable limit and produced no toxic effect. several general and specific roles in plants, and Tolerable limit of aqueous extract of Khaya may exhibit different biochemical and grandifoliola, as investigated and reported by pharmacological actions in different species of Njikam and Njikam [16], showed that the LD of animals when consumed. 50 the plant extract was 5.5 g/kg bodyweight. The

LD50 is over a thousand times more than the In this study, the effect of aqueous 50mg/kg bodyweight shown to be the most extract of Khaya grandifoliola on some efficacious of the three doses administered. disaccharidases activity in iron-deficient weanling rats was investigated. It has been reported 5. CONCLUSION previously that disaccharidases have reduced activity in iron-deficient anemia [3,4]. In this study, the administration of the aqueous extract of Khaya grandifoliola stem bark From Tables (3 and 4), the results showed that, increased the activity of both sucrase and lactase there was significant decrease in the activities of in the intestinal mucosa of iron-deficient weanling the disaccharidases in weanling rats fed iron- rats and 50 mg/kg.bt wt. dose showed to be the

Ajiboye and Oladiji; AJRB, 7(3): 1-7, 2020; Article no.AJRB.60874

most efficacious of all the dose levels. This treatment of anemia. Current hematology investigation furthers strengthen the claim of the reports. 2005;4:436-440. usage of the aqueous extracts of Khaya 6. Smith RE, Aapro MS, Ludwig H, Pintér T, grandifoliola stem bark in the management of Šmakal M, Ciuleanu TE, Chen L, Lillie T, iron-deficient anemia. Glaspy JA. Darbepoetin alfa for the treatment of anemia in patients with active ETHICAL APPROVAL cancer not receiving chemotherapy or radiotherapy: results of a phase III, This study was carried out as approved by the multicenter, randomized, double-blind, Department of Biochemistry Ethical Committee placebo-controlled study. Journal of on the use of laboratory animals at the University Clinical Oncology. 2008;26:1040-1050. of Ilorin with ethical number BCH/SCI/029. 7. Locatelli F, Olivares J, Walker R, Wilkie M, Jenkins B, Dewey C, Gray SJ. Novel DISCLAIMER erythropoiesis stimulating protein for treatment of anemia in chronic renal

insufficiency. Kidney International. 2001; The products used for this research are 60:741-747. commonly and predominantly use products in our 8. Peter EL, Rumisha SF, Mashoto KO, area of research and country. There is absolutely Malebo HM. Ethno-medicinal knowledge no conflict of interest between the authors and and plants traditionally used to treat producers of the products because we do not Anemia in Tanzania: A cross sectional intend to use these products as an avenue for survey. Journal of Ethnopharmacology; any litigation but for the advancement of 2014. knowledge. Also, the research was not funded by 9. Oladiji AT, Jacob TO, Yakubu MT. Anti- the producing company rather it was funded by anaemic potentials of aqueous extract of personal efforts of the authors. Sorghum bicolor (L.) moench stem bark in

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