Therapeutic Evaluation of Combretum Molle (Combretaceae) (Bush Willow) Stembark Extract in Experimental Avian Coccidiosis

South Asian Research Journal of Natural Products

1(3): 1-9, 2018; Article no.SARJNP.43270

Therapeutic Evaluation of Combretum molle (Combretaceae) (Bush Willow) Stembark Extract in Experimental Avian Coccidiosis

M. H. Garba1*, M. L. Sherifat2, A. O. Abdul-Majid1, L. M. Hafsa3, L. J. Bulus1 and G. Yusuf4

1Department of Animal Production Technology, Federal College of Wildlife Management, P.M.B. 268, New Bussa, Niger State, Nigeria. 2Department of Basic Sciences, Chemistry Unit, Federal College of Freshwater Fisheries Technology, P.M.B. 1500, New Bussa, Niger State, Nigeria. 3Department of Basic Sciences, Mathematics Unit, Federal College of Wildlife Management, P.M.B. 268, New Bussa, Niger State, Nigeria. 4Department of Biological Sciences, Federal College of Education, P.M.B.39, Kontagora, Niger State, Nigeria.

Authors’ contributions

This work was carried out in collaboration between all authors. Author MHG designed the study, wrote the protocol and wrote the first draft of the manuscript. Authors LMH and MLS performed the statistical analysis, participated in the writing of the first draft of the manuscript. Authors AOA, LJB and GY managed the analyses of the study and the literature searches. All authors read and approved the final manuscript.

Article Information

DOI: 10.9734/SARJNP/2018/43270 Editor(s): (1) Dr. Prasong Srihanam, Professor, Department of Chemistry, Faculty of Science, Mahasarakham University, Thailand. Reviewers: (1) Kingsley I. Eghianruwa, University of Ibadan, Nigeria. (2) Yamssi Cedric, University of Dschang, Cameroon. Complete Peer review History: http://www.sciencedomain.org/review-history/26079

Received 28 May 2018 Accepted 03 August 2018 Original Research Article Published 03 September 2018

ABSTRACT

Aim: The main thrust of this research work was to assess the potentials of the extract from the stem bark of Combretum molle (identified through ethno-botanical survey) using an appropriate solvent(s) In vivo against coccidial protozoa in experimental animals. Methodology: The stem bark of the plant (Combretum molle) was collected, washed and dried at room temperature. It was then chopped into smaller fragments and pulverized using a grinder. Cold

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

Garba et al.; SARJNP, 1(3): 1-9, 2018; Article no.SARJNP.43270

extraction was performed using 70% methanol which was filtered after 24hrs and then dried using a rotary evaporator. Thirty-five (35), day-old broiler chicks were used. They were kept for acclimatisation period of 2 weeks in the research pen and fed with broilers starter and had unrestricted access to clean water ad-libitum. After the period of acclimatization, the birds were grouped into seven (7) with each group consisting of 5 birds. High standard of hygiene conditions was maintained throughout the course of the research work. Fresh faecal samples from the infected animals were collected. The infective dose of the culture (i.e 74,000 sporulated oocyst/ml) was administered orally using 1ml syringe. The treatment was carried out on three days interval in each group for 21 days (3 weeks). Birds on group 1, 2, 3 and 4 were administered 50 mg/kg bw, 100 mg/kg Bw, 150 mg/kg bw and 200 mg/kg bw C. molle extract respectively. Results: C. molle stem bark extract at a dosage of 50 mg/kg bw, 100 mg/kg bw, 150 mg/kg bw and 200 mg/kg bw in the treatment of broiler chicks infected with Eimeria spp parasites shows reduction in the numbers of oocyst count in each group but the treatment of coccidiosis at a dose of 150 mg/kg bw and 200 mg/kg bw in broiler chicks prove to be more efficient. The assessment of haematological and serum biochemical parameters revealed that C. molle stem bark extract has no pathological effect on the experimental animals. Conclusion: Stembark exract of C. molle could be employed/packaged as phytomedicine against Avian coccidiosis considering its efficacy against the infective oocyst and its potency as a prophylactic agent against the protozoa. Its non-toxicity to the experimental animals is also an added advantage that will further encourage its utilisation.

Keywords: Coccidiosis; Eimeria spp; broiler chicks; ethnobotany; Combretum molle; methanol extract.

1. INTRODUCTION tree up to 10 m high, rarely to 16 m, with a straight regular bole to 1 m girth, of savanna Coccidiosis is a parasitic disease of forest, from Senegal to West Cameroons, and intestinal tract caused by single cell protozoan widespread in tropical Africa. The stems are parasite belonging to genus Eimeria. It causes durable underground and are valuable for house- massive destruction of the epithelial cells, posts. The wood is brownish or yellowish-green, which leads to bloody diarrhoea, reduced very hard and compact, strong and durable, but weight gain and temporary reduction in litter difficult to work. In Sudan, a leafy and stem bark production [1,2]. Seven species have been decoctin is taken to treat fever, jaundice and recognized to infect chicken and each species bacterial infection. Aside of the use of the bark has its own characteristics according to the site for tanning, its extract (stem bark) and leaves of infection, immunogenicity and pathogenicity possessed significant activity against the [3,4]. chloroquine – sensitive Plasmodium falciparum strain NF54 and the leaf extract also totally According to a recent estimate, coccidiosis inhited the enzyme HIV-1 reverse transcriptase may cost the US chicken industry about $127 [7]. million annually [5]. However, due to even lower nutritional efficiency and poor management The intensive use of anti coccidial drugs in practices in Africa, coccidiosis may cost the poultry management has led to the development Africa chicken industry much more annually. of resistance. It, therefore, becomes imperative Likewise, similar losses may occur worldwide to find some natural alternatives which improve particularly the underdeveloped and the growth with either prophylactic or curative developing countries. Thus coccidiosis is effect or both, against coccidiosis without health probably the most expensive and wide spread hazard to a human being. infectious disease in commercial chicken systems. The main thrust of this research work, therefore, is to assess the Anticoccidial Control of coccidiosis mostly depends upon the potentials, Prophylactic effects and acute toxicity chemoprophylaxis by using anti-coccidian drugs of the methanolic stem bark extract of however, managerial skills are also important to Combretum molle (COMBRETACEAE) In vivo, get the maximum anti-coccidian effect of these against coccidial protozoa in experimental Broiler drugs [6]. Combretum molle is a shrub or small chicks.

Garba et al.; SARJNP, 1(3): 1-9, 2018; Article no.SARJNP.43270

2. MATERIALS AND METHODS Group V = infected and not treated (given Normal saline) 2.1 Experimental Site Group VI = not infected and not treated (Normal) Group VII = Infected and treated with the The studies/research work was conducted at the standard drug teaching and research farm and the nutrition and Biochemistry laboratory of the Federal College of 2.6 Drugs Wildlife Management, New-Bussa, Niger State. Toultrazuril obtained from Ministry of Livestock and Fisheries, Minna, Niger State, as an oral 2.2 Experimental Animals R solution (2.5%) under trade name Tolacox .

Day old broiler chicks were purchased at Ibadan, Oyo State. They were kept in the research pen 2.7 Infection of Animals and fed with broilers feed and allowed access to This was carried out as described by Long [8]. clean water ad-libitum. High standard of Briefly, a fresh faecal sample containing Eimeria health/sanitary conditions was maintained oocyst was collected from an infected bird and throughout the course of the research work. placed in a 500 ml capacity beaker containing

distilled water and lightly macerated until 2.3 Plant Materials completely disintegrated and dissolved. The

The ethno-botanical survey was carried out in compound microscope was used to carry out the the surrounding villages namely, Old/New Awuru, oocytes count with subsequent dilution using Koro, Popo, Kere, Lubaruru and Dogongari Mac master chamber until an Oocyst count of villages with the main aim of ascertaining from 74,000 oocytes/ml was attained, which was used the local people (particularly the elderly ones), as an infective dose. the plant species commonly utilised in the The infective dose of the culture (74,000 traditional management of coccidiosis. Part(s) Oocytes/ml) was administered orally using a 1 ml utilised the method of preparation and period of insulin syringe which was introduced intra-crop. harvest were also enquired from the The treatment thereafter, commenced on day six interviewees. The identity of the plant was (6) (prepatent period) in each group as indicated confirmed by Mr Musa Idris in the Department of in section 2.5.1 for 21 days (3 weeks). Forestry, Federal College of Wildlife

Management, New Bussa, Nigeria. 2.8 Prophylactic Activity Test

2.4 Plant Preparation The test for prophylactic activity was done as described by Jocelyn et al. [9] and Ogbadoyi et The stem bark of the plant Combretum molle al. [10]. (Bush willow) was washed with clean water and dried at room temperature and then chopped into 2.9 Parasitological Examination (Oocytes smaller fragments and pulverised using a Count) grinder. Cold extraction was performed using 70% methanol and rotary evaporator was to A fresh faecal sample in each of the treatment remove the solvent. groups I – VII (except VI) was collected. This was lightly macerated and subsequently washed and 2.5 Pharmacological Studies filtered through gauze and the final clear liquid sample containing the oocytes was poured into a 2.5.1 Anti coccidian effect beaker containing normal saline and kept for oocytes count using a compound microscope. A total number of thirty-five (35) broiler chickens The count was conducted at three days interval. were grouped into seven (7) treatments with each treatment consisting of five (5) chickens as 2.10 Haematology thus: On day 21, blood samples from each treatment Group I = 50 mg/kg bw group were collected into labelled Group II = 100 mg/kg bw Ethylenediamine tetra acetic acid (EDTA), Group III = 150 mg/kg bw treated tubes for haematological analysis. The Group IV = 200 mg/kg bw determination of the haematological parameters

Garba et al.; SARJNP, 1(3): 1-9, 2018; Article no.SARJNP.43270

was carried out using an automated haemato increases, there is a corresponding increase in analyser (SpectruMedix 329LX) at the general the activity of the extract as shown in Fig. 3.1. At hospital Minna, Niger State, Nigeria. the concentration(s) of 100, 150 and 200 mg/Kg bw, it became obvious that activity of the extracts 2.11 Serum Biochemistry in terms of oocytes count is higher than the standard drug. Blood samples were also collected into tubes without anticoagulant and the serum analysis on 3.1 Prophylactic Activity Test Blood glucose, alkaline phosphatase (ALP), Serum albumin, Total protein, Cholesterol and Administration of a curative dose of 200 mgkg-1 Blood urea was carried out using Radox analysis bw of the stem bark extract of Comberetum molle kit. to five different groups of healthy chickens for five consecutive days prior to infection appears 2.12 Acute Toxicity Studies to protect them from infection. The animals were observed not to have come up with the parasites Doses of 600, 900, 1200 and 1500 mg kg-1bw of after infection. The prophylactic activity displayed the extract were orally administered to non- by the extract at a curative dose of 200 mg/kg bw infected chicks and were observed for 72 hours. is also an added advantage in the usage and progressive intensification of research to identify 2.13 Statistical Analysis and develop the lead compound from the plant (Fig. 3.2). The data collected were subjected to one-way analysis of variance (ANOVA) in a completely One advantage that a drug may have over others randomized design (CRD) arrangement. The also lies in its haemopoietic and immuno- significant means were separated and compared modulatory role. It could be observed that there using Duncan multiple range tests (DMRT). is no significant difference (P ≤ 0.05) in the haemoglobin (Hb), packed cell volume (PCV), 3. RESULTS mean corpuscular haemoglobin concentration (MCHC) and the red blood count (RBC) The efficacy of the extract appeared to be between the normal control and the groups concentration dependent. As the concentration administered various doses of the extract.

30 50mg/kgbw ) 25 3

0 100mg/kgbw 1

x 20 ( 150mg/kgbw t n

u 15 200mg/kgbw o c

Nagative control t

s 10

y Standard drug c

o 5 O 0 3 6 9 12 15 18 21 -5 Days

Fig. 3.1. Efficacy of the extract administer at dose of 50 mg, 100 mg, 200 mg, negative control and standard drug

Garba et al.; SARJNP, 1(3): 1-9, 2018; Article no.SARJNP.43270

0.25

0.2 3 0 1

t 0.15 n u o C

200mg/kgbw t

s 0.1

y prophylactic dose c o O 0.05

0 1 2 3 4 5 7 9 11 13 15 Days

Fig. 3.2. Prophyactic activity of Combretum molle in experimental birds

While the higher concentration (P ≤ 0.05) in the observed in the animals administered the extract Platelet counts PLC), Total white blood t h a n the normal control, which is a clear count (TWB), RDW, Lymphocytes counts, indication of immuno-stimulatory/modulatory Monocytes,Eosinophil and basophil counts was activity of the extract [11] (Fig. 3.3).

120

100

positive control 60 50mg/kgbw 100mg/kgbw

40 150mg/kgbw 200mg/kgbw

-20

Fig. 3.3. Haematological parameters of broilers chickens treated with Combretum molle HB = Haemoglobin, PCV = Packed Cell Volume, RBC = Red Blood Count, TWBC =TOTAL White blood Count, MCV = Mean cell volume, MCHC = Mean corpuscular haemoglobin concentration, MCH = Mean corpuscular haemoglobin, N=Neutrophils, L=Lymphocytes, M=Monocyte, E=Eosinophils, B=Basophils

Garba et al.; SARJNP, 1(3): 1-9, 2018; Article no.SARJNP.43270

250

200

150 positive control

50mg/kgbw

100 100mg/kgbw

150mg/kgbw

200mg/kgbw 50

-50

Fig. 3.4. Serum biochemical characterisctics of broilers treated with Combretum molle ALP = Alkaline phosphatase

Table 3.1. Effects of acute administration of various doses of the crude extract to healthy chickens

Dosage No of animals T/D Observations Distilled H20 or 4 4/0 No sign of toxicity, animals remained active Normal Saline even after the administration. 300 mgkg-1bw 4 4/0 No sign of toxicity, animals remained active even after the administration. 600 mgkg-1bw 4 4/0 Looked a bit depressed, the breathing was slow and remained Sluggish for a short while became normal again. 900 mgkg-1bw 4 4/0 Sluggishness was observed, the breathing was slow and there was the closing of the eyes and the feathers stood erect but conditions returned to normal after about 24h. 1200 mgkg-1bw 4 4/0 Sluggishness was observed, the breathing was slow and there was the closing of the eyes and the feathers stood erect but conditions returned to normal after about 24h. 1500 mgkg-1bw 4 4/0 Sluggishness was observed, the breathing was slow and there was the closing of the eyes and the feathers stood erect but conditions returned to normal after about 48h. T/D = Number of deaths recorded from the total number of animals in each group

4. DISCUSSION 200 mg/kgbw (Fig. 3.1) this is a confirmation of the previous reports that organism are gradually Findings made from this study showed higher developing resistance to most of the drugs efficacy of the crude extract of Combretum molle currently in use. It could also be observed that compared to the standard drug Toultrazuril the efficacy of the extract is concentration R (Tolaclox ) at a concentration of 100, 150 and dependent. What this interesting finding reveals

Garba et al.; SARJNP, 1(3): 1-9, 2018; Article no.SARJNP.43270

is the fact that, further purification of this extract, probably due to successive replacement of the may possibly yield a fraction that will be more protein albumin by the paraproteins in the groups efficacious at a lower dosage, than the currently administered the extract. It is pertinent to state available drugs. This will certainly open a new that, there is no significant difference (P ≤ 0.05) window of opportunity for the rural farmers to in the cholesterol, urea, glucose and alkaline have a better alternative that will be less costly, phosphatase concentration between the normal readily available and more accessible. Though control and the groups administered the extracts. we cannot say with certainty, the This observation will certainly go a longway to pharmacokinetics of this extract, this interesting show the relative safety of chronic administration result might not be unconnected with some of this extract. phyto-components reviewd by Sudipta et al. [12] which include: CombreteneA, Combretene B, On the acute toxicity of this extract, Table 3.1 2α,3β-,6β--trihydroxy-23-, galloylolean-12-en-28- clearly indicates that it is relatively safe at a dose oate, Combregenin, Arjungenin, Arjunglucoside of 1500 mg/kgbw, this corroborates the findings I, Combreglucoside, Sericoside, Mollic Acid, of Ademola and Eloff [36], that found it to be safe Mollic acid 3β-O-glucoside, Mollic acid 3β--O- on acute administration to albino mice at a dose arabinoside, Ellagitannin and Punicalagin of 849–900 mg/KgBw. identified to be associated with this plant [13,14,15,16,17,18,19,20,21,22,23,24,25]. These 5. CONCLUSION phyto-components were shown to individually or in synergy with some other molecules display Taken the results from this study together, the high antiprotozoal, antifungal, antihelmintic, anti- methanol stem bark extract of Combretum molle inflamatory and anti molluscidal activities against appears to be more efficacious than the range of pathogenic organisms [12]. Therefore, conventional drug Toultrazuril (TolacoxR) at the the high anti-coccidial effect might be as a result doses of 100, 150 and 200 mg/KgBw. Its of similar or interplay of the mode of actions Prophylactic potentials is also a relief and exhibited in other pathogens by these phyto succour to the poor rural farmers that suffers chemicals. most dilapidating damages caused by this disease. The low chronic toxic effect (except This novel prophylactic property displayed by this paraproteinemia) and its non-acute toxicity even plant extract has not been displayed by plants at a dose of 1500 mg/KgBw exhibited by this such as Aloe vera, Aloe spicata, Myrothamnus extract will certainly encourage its deployment in flabellifoilius, Lannea stullmannii, Capsicum the management of this disease. However, annum (Pepper), Parinaria curatellifolia, Albizia further research on the potency of the partially gummisera, Albizia adianthifolia and Soot purified (fractionated) extract will shade more investigated by Marizvikuru et al. [26]. Though light on the active component and its possible Lannea stullmannii was found to possess a mode of action. similar activity [27]. It has to be added into the feed at a high ratio of 1:4 which may not bring ETHICAL APPROVAL down the cost of production.

An interesting observation made in relation to the Authors hereby declare that "principles of plasma protein concentration due to the Laboratory animal care" (nih publication no. 85- administration of this extract is the significant 23, revised 1985) were followed, as well as increase (P ≤ 0.05) across all the groups Specific national laws where applicable. All administered the extract compared with the Experiments have been examined and approved normal control (Fig. 3.4). Since the animals were by the appropriate ethics committee. allowed access to drinking water ad libitum, this condition may not be attributed to the COMPETING INTERESTS dehydration but rather paraproteinemia that may probably be induced by the extracts as a result of Authors have declared that no competing their ability to induce B- lymphocytes or plasma interests exist. cells to produce these monoclonal proteins that exist as heavy chain subtypes (IgG, IgA, IgG, REFERENCES and less commonly IgD or IgE) and light chain subtypes (kappa or lambda) [28]. The exact 1. Razzaq A, T Ali, A Saghir, Arshad S, opposite of protein concentration was observed Cheema A. Trainingneeds assessment of with the serum albumin concentration (Fig. 3.4) chicken farmers in tehsil Faisalabad.

Garba et al.; SARJNP, 1(3): 1-9, 2018; Article no.SARJNP.43270

Journal of Animal and Plant Science. clinical trial. Journal of Clinical 2011;21:629-631. Pharmacology and Therapy; 2018. 2. Dalloul RA, HS Lillehoj. Recent advances DOI: 10.1111/jcpt.12684 in immune modulation and vaccination 12. Sudipta R, Dilip G, Rabinarayan A, Rajiv strategies against coccidiosis. Avian R. Combretum (Combretaceae): Biological Diseases. 2005;49:1-8. activity and phytochemistry: A Review. 3. Akhtar M, Anwar MI, Iqbal Z, Muhammad Indo American Journal of Pharmaceutical F, Awais MM, Haq AU, Sawicka EH. Research. 2014;4(11):5226–5299. Immunological evaluation of two local ISSN NO: 2231-6876 isolates of Eimeria tenella gametocytes 13. Bahar A, Tawfeq AAH, Passreiter CM, against coccidiosis in chicken. Pakistani Jaber SM. Combretene A and B, two new Veterenary Journal. 2012;32:77-80. triterpenes from Combretum molle. 4. Williams RB. Epidemiological aspects of Pharmacy and Biology. 2008;42(2):109- the use of live anticoccidial vaccines for 113. chickens. Internatinal Journal of 14. Kemvoufo PB, Barboni L, Teponno RB, Parasitology. 1998;28:1089-109. Mbiantcha M, Nguelefack TB, Hee-Juhn P, 5. Chapman HD. A landmark contribution to Kyung-Tae L, Tapondjou LA. chicken science prophylactic control of Polyhydroxyoleanane-type triterpenoids coccidiosis in chicken. Poulty Science. from Combretum molle and their anti- 2009;88:813-815. inflammatory activity. Phytochemistry. Letter. 2008;1(4):183-187. 6. Tewari AK, Maharana BR. Control of 15. Wang LQ, Wu MM, Liu JP, Li Y, Hua Y, chicken coccidiosis: Changing trends Wang YY, Li XY, Chen YG, Wang JH. Five Journal of Parasitic Dieases. 2011;35:10- new diarylpropan-1-ols from Combretum

70. yunnanense. Planta Medicine. 2011; 7. Maroyi A. Cmbretum molle R.Br.ex G.Don. 77(16):1841-1844. In: Schmelzer, G.H. & Gurib-Fakim, A. 16. Jossang A, Seuleiman M, Maidou E, Bodo (Editors). Prota. Medicinal plants/plantes B. Pentacyclictriterpenes from Combretum medinales 2. PROTA, Wageningen, nigricans. Phytochemistry. 1996;41(2): Netherlands. 2012;11(12). 591-594. (Accessed: 23 March 2018) 17. Asres K, Bucar F, Knauder E, Yardley V, 8. Long PL, Joyner LP, Millard BJ. A guide to Kendrick H, Croft SL. In vitro antiprotozoal laboratory techniques used in the study activity of extract and compounds from the and diagnosis of Av.coccidisis. Folia stem bark of Combretum molle. Veterenary. Letter. 1976;(6):201-217. Phytotherapy Research. 2001;15(7):613- 9. Jocelyn AK, Sergio R, Navarro-Vela AM, 617. Raffy D, Marco V, Hildegund E, David D, 18. Pegel KH, Rogers CB. The Helen R, Louis HN, Abela-Ridder B, characterisation of mollic acid 3β-D- Deborah B. Pre-exposure rabies xyloside and its genuine aglycone mollic prophylaxis: A systematic review Bulletin acid, two novel 1α-hydroxycycloartenoids of World Health Organ. 2016;95(3):210– from Combretum molle. Journal of 219. Chemical Societyof Perkin Transactions. DOI: 10.2471/BLT.16.173039 1985;1:1711-1715. 10. Ogbadoyi EO, Garba MH, Kabiru AY, 19. Rogers CB, Coombes PH. Mollic acid and Mann A, Okogun JI. Therapeutic its glycosides in the trichome secretions of evaluation of Acacia nilotica (Linn) stem Combretum petrophilum. Biochemical. bark extract in experimental African System and Ecology. 2001;29(3):329-330. trypanosomiasis. International Journal of 20. Rogers CB. Isolation of the 1α- Applied Research in Natural Products. hydroxycycloartenoid, mollic acid α-l- 2011;4(2):11-18. arabinoside from Combretum edwardsii 11. Khazdair MR, Alavinezhad A, Boskabady leaves. Phytochemistry. 1989;28(1):279- MH. Carvacrol ameliorate haematological 280. parameters, oxidant/ antioxidant 21. Ganzera M, Ellmerer-Müller EP, Stuppner biomarkers and pulmonary function tests in H. Cycloartane triterpenes from patients with sulphur mustard-induced lung Combretum quadrangulare. Phyto- disorders: A randomized double-blind chemistry. 1998;49(3):835-838.

Garba et al.; SARJNP, 1(3): 1-9, 2018; Article no.SARJNP.43270

22. Roy R, Singh RK, Jash SK, Sarkar A, Journal of Ethnopharmacology. 2007; Gorai D. Combretum quadrangulare 110(1):56-60. (Combretaceae): Phytochemical 26. Marizvikuru Mwale, Evison Bhebhe, constituents and biological activity. Indo Michael Chimonyo, Tinyiko Edward American Journal of Pharmaceutical Halimani. Use of Herbal Plants in Poultry Research. 2014;4(8):3416-3430. Health Management in the Mushagashe 23. Facundo VA, Andrade CHS, Edilberto RS, Small-Scale ommercial Farming Area in Braz-Filho R, Hufford CD. Triterpenes and Zimbabwe. International Journal of Applied flavonoids from Combretum leprosum. Research in Veterinary Medicine. 2005; Phytochemistry. 1993;32(2):411-415. 3(2):163-170. 24. Adnyana IK, Tezuka Y, Awale S, Banskota 27. Gueye FHE. Diseases in village chickens: AH, Tran KQ, Kadota S. QuadranosidesVI- Control through ethno-veterinary medicine. XI, six new triterpene glucosides from the Centre for Research and Information on seeds of Combretum quadrangulare. Low-External-Input and Sustainable Chemical Pharmacology. Bulletin. 2000; Agriculture (ILEIA) Newsletter. 2000;13: 48(8):1114-1120. 20–21 25. Angeh JE, Huang X, Sattler I, Swan GE, 28. Richard A. Rison, Said R. Beydoun. Dahse H, Härtl A, Eloff JN. Antimicrobial Paraproteinemic neuropathy: A practical and anti-inflammatory activity of four review, BMC Neurology. 2016;16:13. known and one new triterpenoid from DOI: 10.1186/s12883-016-0532-4 Combretum imberbe (Combretaceae). PMCID: PMC4731930 ______© 2018 Garba et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Peer-review history: The peer review history for this paper can be accessed here: http://www.sciencedomain.org/review-history/26079