Urolithiasis
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The aim of this study was to measure the effects of the aqueous extract of leaves of Adansonia digitata (AAD) in vitro on calcium oxalate (CaOx) nucleation and aggregation [by spectrophotometric time ...

The aim of this study was to measure the effects of the aqueous extract of leaves of Adansonia digitata (AAD) in vitro on calcium oxalate (CaOx) nucleation and aggregation [by spectrophotometric time course measurements of optical density at 620 nm (OD620)]. For measuring calcium oxalate crystallization inhibitor activity agar gel model, and in vivo on experimentally induced CaOx urolithiasis in male Wistar rats. CaOx urolithiasis in rats was induced by intraperitoneal (i.p.) injection of sodium oxalate (NaOx) (7 mg/100 g/day for 7 days). AAD was administered orally (200 mg/kg/day for 7 days). Urine volume, pH, body weight, kidney weight (wet and dry), serum and urine level of creatinine, urea, magnesium (Mg2+), calcium (Ca2+) were evaluated on day 7. In addition, histopathological changes in kidney and oxalate in urine and kidney were evaluated. The results revealed that AAD inhibited the rate of crystal nucleation (SN) and aggregation (SA) and showed inhibitory activity on CaOx crystallization. The histopathological examination of kidneys revealed that AAD significantly reduced the incidence of CaOx crystal deposition. In addition, AAD significantly increased urinary excretion of Mg2+ along with a decrease of oxalate excretion. In conclusion, the aqueous extract contains potent antiurolithiatic substances which warrant further evaluation.

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Urolithiasis Presentation Transcript

  • 1. Pharmacological Evaluation of Antiurolithic Activity of Leaves of Adansonia digitata Linn. SUBMITTED BY Mr. Swaroop Singh SUPERVISOR Prof. (Dr.) Neelam Balekar College of Pharmacy, IPS Academy, Rajendra nagar, A.B. Road, Indore-452012 2010-2011 1
  • 2. 1. INTRODUCTION  Urolithiasis denotes stones originating anywhere in the urinary tract, including the kidney and bladder (Pearle & Nakada, 2009).  Overall male to female ratio of 2.4:1 increased from 1977 (1.86:1) to 2006 (2.7:1) (Knoll et al., 2011).  Recurrence rate (10 to 20% within 1 to 2 years, 35% within 5 years, & 60% within 10 years) (Berner et al., 2011).  Types of kidney stones (Dipiro et al., 1997) • Calcium stones (CaOx and CaP)- 80% • Uric acid- 9% • Struvite-10% • Others- 1% College of Pharmacy, IPS Academy Fig. 1. Stones in kidney 2
  • 3. 1.1. PATHOGENESIS  Urine is a complex fluid that contains a number of minerals, inhibitors and promoters of crystallization.  Crystallization can not occurs whether promoters and inhibitors are present or absent, if a state of supersaturation does not exist. Fig. 2. Scheme of the precess of stone formation (Tsujihata, 2005). College of Pharmacy, IPS Academy 3
  • 4. 1.2. Possible series of events of the normal and pathological crystallization in urine (adopted from ‘Oxford Textbook of Clinical Nephrology’). Fig. 3. Normal crystallization in urine. Fig. 4. Pathological crystallization in urine. College of Pharmacy, IPS Academy 4
  • 5. 1.3. Risk factor associated with kidney stones  Low urine volume  Hypercalciuria (most common)  Hypocitraturia Primary defect in renal tubular reabsorption of calcium  Hyperoxaluria Ion activity product Supersaturation Excretion of Calcium in urine Plasma level of calcium Renal hypercalciuria PTH Resorptive hypercalciuria Fig.5. Types of hypercalciuria College of Pharmacy, IPS Academy Production of vit. D3 in kidney Calcium transport from lumen of gut Absorptive hypercalciuria 5
  • 6. 2. MANAGEMENT OF KIDNEY STONES Therapeutic approaches  Removal of stones (surgical management & MET)  Lowering supersaturation (fluid intake & dietary modification)  Prevent crystal retention in kidneys  Reduction of renal oxidative stress 2.1. SURGICAL MANAGEMENT (Pearle & Nakada, 2009) 1) ESWL 2) PCNL 3) URS (flexible ureteroscopy) College of Pharmacy, IPS Academy 6
  • 7. Cont… 2.2. MEDICAL EXPULSIVE THERAPY (Pearle & Nakada , 2009)  A new clinical approach  Urethral smooth muscle relaxation  CCB & alpha-1 receptor blockers 2.3. FLUID INTAKE AND DIETARY MODIFICATION  High fluid intake in order to maintain a urine output of at least 2 L/day  Reduced supersaturation and dilutes promoters of CaOx crystallization  Restriction of animal protein diet  Restriction of Na intake (2-3gm)  No restriction of calcium College of Pharmacy, IPS Academy 7
  • 8. Cont… 2.4. PHARMACOLOGICAL TREATMENT (Dipiro et al., 1997)  Diuretics  Alkali Potassium citrate, potassium magnesium citrate  Pyridoxine 2.5. LIMITATION  Persistent of stone fragments cause renal injury & an increase in stone recurrence with ESWL & development of diabetes and hypertension on long term use (Butterweck & Khan 2000).  Long term use of thiazide causes K depletion (hypokalemia), Fatigue, dizziness, impotence & musculoskeletal symtoms. College of Pharmacy, IPS Academy 8
  • 9. 3. PLANT PROFILE 3.1. BIOLOGICAL SOURCE  Botanical name : Adansonia digitata L.  Family : Bombacaceae 3.2. PHARMACOLOGICAL ACTIVITIES OF LEAVES OF PLANT  Anti-ulcer activity (Karumani et al., 2008)  Antimicrobial activity (Oloyede et al., 2010)  Antioxidant activity (Vetuani et al., 2002)  Antiviral activity (Hudson et al., 2000 ; Ananil et al., 2000)  Diuretic activity (Kirubha et al., 2006)  Anti-inflammatory activity (Selvarani & Hudson, 2009)  Antisickling activity (Adesanya et al., 1988) College of Pharmacy, IPS Academy 9
  • 10. 3.3. TRADITIONAL USES OF LEAVES OF Adansonia digitata (Sibibe & Willians, 2002)  Insect bite & gunia worm sores & otitis  Diseases of the bladder and urinary tract  Antiasthmatic  Antipyretic  Fatigue  Dysentery  Opthalmia  Astrigent  Diaphoretic  Tonic College of Pharmacy, IPS Academy Fig.6. Leaf of Adansonia digitata. 10
  • 11. 3.4. PHYTOCHEMICAL CONSTITUENT OF LEAVES OF PLANT  Excellent source of amino acids (Yazzie et al., 1994)  Carbohydrate , fat, protein and fibre (Caluwe., 2010)  Antioxidants such as catechins and adansonia flavonosides (Maranz et al., 2007; SEPASAL database., 2007; Kunkel ., 1979)  Minerals (iron, zinc, calcium, magnesium, manganese, copper, phosphorus & sodium) (Glew et al., 1997)  Provitamin A along with thiamine, riboflavin &niacin (Caluweet al., 2010)  Mucilage (Woolfe et al., 1977)  Alkaloids & Steroids (Oloyede et al., 2010)  Tannis (Oloyede et al., 2010)  Cardio active glycosides (Oloyede et al., 2010) College of Pharmacy, IPS Academy 11
  • 12. 4. OBJECTIVE  The objective of present study to assess the effectiveness of leaves of Adansonia digitata L., a medicinal plant used to treat kidney and bladder disease as traditionally, as a prophylactic agent against CaOx stones by in vitro and in vivo study.  The present study shall undertake • Collection of plant material from local area. • Identification and authentication of the plant from Botanical survey of India. • Preparation of suitable extracts of selected plant material. • Evaluation of the extract for antiurolithic activity by in vitro and in vivo study. College of Pharmacy, IPS Academy 12
  • 13. 4.1. RATIONALE  No satisfactory drug to use in clinical therapy for the prevention or the recurrence of stones.  Traditional claim of the plant(Sidibe & Willians, 2002; Caius, 2003).  Absence of literatures on the systematic investigation of antiurolithic activity.  Data from In vitro and In vivo & clinical trials reveal that phytotherapeutic agent could be useful in the management of urolithiasis. In this regards many plant have been used to treat stones like Dolichous biflorus, Berginia ligulata, Tribulus terrestris, Orthosiphon grandiflorus, Phylanthus niruri (Butterweck & khan, 2000).  Diuretic activity (Kirubha et al., 2006) & antioxidant activity (Vetuani et al., 2002).  Antiurolithic activity of L-arginine amino acid (Pragasam et al., 2005). College of Pharmacy, IPS Academy 13
  • 14. 5. PLAN OF WORK PLAN OF WORK Minor project Phytochemical investigations and Major project pharmacognostic Detailed pharmacological investigations of antiurilithic activity Collection of plant Authentication of plant material Preparation of aqueous extract of leaves In vivo study Sodium oxalate induced urolithiasis model in male rats In vitro study A gel model for measuring crystallization inhibitor activity Nucleation and aggregation assay Fig.7. Plan of work College of Pharmacy, IPS Academy 14
  • 15. 6. EXPERIMENTAL WORKS AND RESULTS 6.1. PLANT MATERIALS AND EXTRACTION PROCEDURE  Leaves of Adansonia digitata were collected from MHOW, Indore & authentication of plant was done in BSI, Pune (a voucher specimen number BSI/WC/Tech./2011/900A).  Aqueous extract was prepared by decoction method (temperature 90-95ºC) and was dried at 37ºC. Yild was found 15.8 % (Pauly, 2001). 6.2. AGAR GEL MODEL (Schneider et al., 1983) Fig.8. Diagram of punch-hole pattern (all dimensions in mm) of agar gel model College of Pharmacy, IPS Academy 15
  • 16. 6.2.1. Protocols For Agar Gel Model Table 1:Protocols for agar gel model. Group First method Second method Modifier (70 µL) Modifier (conc. in agar gel) Control Distilled water Distilled water Standard Magnesium chloride (100mM) Magnesium chloride (100mM) Trisodium citrate (100mM) HA extract (1mg/mL) HA extract (1mg/mL) HA extract (10mg/mL) Test Trisodium citrate (100mM) HA extract (10mg/mL) College of Pharmacy, IPS Academy 16
  • 17. 6.2.2. Results of Agar Gel Model Crystallization streak’s Pattern of CaOx crystal produced by starter solution of 0.3 M CaCl2 and ammonium oxalate in agar gel with addition of a. distilled water. b. 200 mM of MgCl2. c. 1 mg/mL AAD. d. 10 mg/mL of AAD. e. 25 mg/mL of AAD. f. distilled water. g. 200 mM of MgCl2. h. 100 mM of MgCl2. i. 1 mg/mL of AAD. j. 10 mg/mL of AAD; n=6 Fig.9. Pattern of streak of caox cyrstal in agar gel College of Pharmacy, IPS Academy 17
  • 18. 6.3. NUCLEATION AND AGGREGATION ASSAY (Kulaksizoglu et al., 2008 & Hennequin et al.,1993)  The nucleation and aggregation of CaOx were studied at pH 5.7 using turbidimetric measurment at 620 nm of suspensions produced by mixing calcium chloride and sodium oxalate (4 mM & 0.5 mM respectively).  Stock solutions (pH 7.5) • Calcium chloride (8 mM, containing 200 mM NaCl & 10 Mm sodium acetate). • Sodium oxalate (1 mM, ontaining 200 mM NaCl & 10 Mm sodium acetate). • Trisodium citrate (0.30 mM) & test solutions (0.2, 2.0 & 20.0) in calcium chloride stock solution.  All experiments with modifiers of CaOx crystallization were performed at assay conc. of 4 mM CaCl2, 0.5 mM Na2C2O4, 200 mM NaCl, 10 mM sodium acatate, 0.1, 1.0, 10.0 mg/mL of extract, pH 5.7. College of Pharmacy, IPS Academy 18
  • 19. SN SA Equilibrium 0.08 0.07 OD (620 nm) 0.06 0.05 0.04 0.03 0.02 0.01 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Time (min) Fig. 10. Time Course Measurements of OD at 620 nm At Standard Condition of CaOx Crystallization College of Pharmacy, IPS Academy 19
  • 20. 6.3.1. Results of Nucleation and Aggregation Assay. Table 2: Effect of extract on calcium oxalate crystallization. Parameters Control tmax (min) Std Test 0.1mg/mL 1.0 mg/mL 10.0 mg/mL 7.96 ± 0.31 12.47 ± 0.54 12.38 ± 0.79 13.22 ± 0.38* 23.68 ± 1.69*** SN ( 10-2) 1.05 ± 0.04 0.36 ± 0.03** 0.41 ± 0.02* 0.23 ± 0.04*** % Inhibition of SN - 65.12 ± 3.70 57.71 ± 5.40 60.42 ± 2.20 77.93 ± 4.10 SA ( 10-3) 1.24 ± 0.05 0.64 ± 0.06** 0.70 ± 0.02 0.57 ± 0.05*** 0.63 ± 0.05* % Inhibition of SA - 48.42 ± 5.54 43.41 ± 2.04 53.03 ± 4.95 49.19 ± 4.08 0.52 ± 0.08 *P < 0.05,** P < 0.01,***P < 0.001 vs control (Kruskal-Wallis variance analysis); n=6-9, College of Pharmacy, IPS Academy SEM 20
  • 21. 6.4. ANIMAL MODEL OF SODIUM OXALATE INDUCED UROLITHIATIC IN MALE RATS (Gupta et al., 2006; Khan et al., 1981., Ramesh et al., 2010) Table 3: Treatment Schedule Groups Treatment (daily for7 days) Normal control 0.9% saline (1 mL/100g, orally & i.p.) Urolithiactic group NaOx (7 mg/100g, i.p.) + 0.9% saline orally (1 mL/100g) Standard group NaOx (7 mg/100g, i.p.) + potassium citrate monohydrate (0.25g/100g, orally) Treated group NaOx (7 mg/100g, i.p.) + aqueous extract (200mg/kg, orally) Urine volume, pH, body weight, kidney weight (wet and dry), serum and urine level of creatinine, urea, magnesium, and calcium were evaluated on day 7. In addition, histopathological changes in kidney and oxalate in urine and kidney were evaluated. College of Pharmacy, IPS Academy 21
  • 22. 6.4.2. Results of Sodium Oxalate Induced Urolithiasis Table 4: Effect of ADD on serum chemistry in control and experimental animals Parameters Urolithiactic PCi treated/ ADD Treated Control Standard group group Control (mg/dl) Ca++ 10.44 ± 2.36 11.99 ± 0.065 10.26 ± 2.68 12.68 ± 4.96 Mg++ 2.48 ± 0.21 1.63 ± 0.21*** 2.76 ± 0.19### 2.90 ± 0.090### Urea 8.19 ± 1.03 27.36 ± 5.59*** 14.21 ± 4.75### 11.78 ± 1.69### Creatinine 0.59 ± 0.19 2.13 ± 0.77** 0.89 ± 0.13### 0.85 ± 0.07## Data are expressed as mean ± SD of 5 animals per group; * p< 0.05, versus control group; # p< 0.05, ## p< 0.01, ### p< ** p< 0.01, *** p< 0.001 0.001 versus urolithiatic control; one-way analysis of variance, ANOVA followed by Newman–Keuls test for multiple comparisons College of Pharmacy, IPS Academy 22
  • 23. Table 5: Effect of AAD on urinary chemistry in control and experimental animals Parameters Control group Urolithiactic PCi treated/ ADD treated control standard group group Vol. (ml/100 g) 6.34 ± 4.11 4.51 ± 0.48 7.78 ± 1.32 6.02 ± 2.06 pH 8.78 ± 0.24 7.30 ± 0.84* 8.97 ± 0.97# 8.85 ± 0.22# Ca++ (mg/dl) 28.96 ± 4.99 8.31 ± 1.40*** 4.84 ± 3.47*** 3.35 ± 0.89*** Mg++ (mg/dl) 2.50 ± 0.13 2.09 ± 0.10 2.80 ± 0.17# 3.35 ± 0.43### Ox (µg/dl) 23.94 ± 0.84 29.48 ± 1.15*** 23.83 ± 0.86### 24.35 ± 0.46### Urea (mg/dl) 1686 ± 667.7 543.3 ± 174.8* 1529.7 ± 261.5# 1830.1 ± 248.1# Creatinine 57.04 ± 15.05 16.92 ±1.43** 55.27 ±9.44## 41.50 ±4.67# (mg/dl) College of Pharmacy, IPS Academy 23
  • 24. Table 6: Effect of AAD on kidney weight and chemistry in control and experimental animals Parameters Control Urolithiactic PCi treated/ ADD Treated Control Standard group group Wet Weight (mg/100 g) 385.00 ± 17.65 674.60 ± 62.87*** 474.70 ± 75.02### 527.80 ± 36.19### Dry Weight (mg/100 g) 104.90 ± 9.46 127.80 ± 9.79** 95.09 ± 12.73## 100.60 ± 13.47## Width (mm) 7.40 ± 0.89 11.80 ± 0.83*** 9.00 ± 0.70### 9.80 ± 0.83## length (mm) 13.80 ± 1.30 19.00 ± 0.70** 14.00 ± 3.08## 14.80 ± 2.77## Ox (µg/100 mg) 3.642 ± 0.22 6.12 ± 1.96* 4.41 ± 0.57# 3.59 ± 0.45# Ca++ (mg/100 mg) 0.49 ± 0.22 1.34 ± 0.48** 0.75 ± 0.16# 0.76 ± 0.13# College of Pharmacy, IPS Academy 24
  • 25. Renal tissue of a. Control group’s rats showing normal kidney parenchyma with glomerulus, peroximal and distal convoluted tubules with normal brush-broder and intact glomeruli (×100). b. Control group’s rats showing no sign of crystallization (×400) . c Urolithiatic rats showing focal areas of necrosis, infiltration of inflammatory cells and atrophy glomerulus (×100). d. Urolithiatic rats showing deposition of numerous crystals and presence of casts and extensive haemorrhages in collecting tubules (×400). e. Urolithiatic rats showing deposition of salt of calcium, extensive atrophy of tubular lumen as well as dialated tubules (×400). f. Urolithiatic rats showing extensive haemorrhages and infiltration of inflammatory cells (×400). g. PCi treated rats showed moderate haemorrhage and absence of CaOx crystals (×100). h. PCi treated rats showing regeneration of tubules with large hyperchromic nuclei (×400). i. AAD treated animals showing highly regenerating tubules with normal brush-broder and absence of CaOx crystals (×100). j. AAD treated animals showing no deposition of crystals with rare tissue injury (×400). Fig .11. Light microscopic of H & E stained kidney section College of Pharmacy, IPS Academy 25
  • 26. 7. CONCLUSIONS 7.1. CONCLUSIONS  Extract of leaves of Adansonia digitata could act as antilithic agent , by inhibiting nucleation and aggregation of calcium oxalate crystals.  High binding of extract with oxalate could prevent dietary absorption of oxalate form intestine.  In conclusion, based on our present results it seems that AAD contains several active compounds which in a multifunctional/synergistic approach act as antiurolithic. The mechanism underlying this effect is mediated possibly through an antioxidant, antimicrobial, and/or anti-inflammatory activities contained in ADD and presence of inhibitors of CaOx crystallization like magnesium and acidic amino acids. In vitro and in vivo experiments confirm that AAD could be beneficial in the management of CaOx stone disease. College of Pharmacy, IPS Academy 26
  • 27. 9. REFERENCES 1. 2. 3. 4. 5. 6. Ananil, K, Hudson, JB, Souzal, CD, Akpaganal, K, Tower, GHN, Arason, JT & Gbeassor, M 2000, ‘Investigation of medicinal plants of togo for antiviral and antimicrobial activities’, Pharmaceutical Biology, vol. 38, no. 1, pp. 40-45. Atmani, F, Slimani, Y, Mimouni, M & Hacht, B 2003, ‘Prophylaxis of calcium oxalate stones by Herniaria hirsute on experimentally induced nephrolithiasis in rats’, BJU International, vol. 92, pp. 137-140. Brener, ZZ, Winchester, JF, Salman, H & Bergman, M 2011, ‘Nephrolithiasis: evaluation and management’, Southern Medical Journal, vol. 104, no. 2, pp. 133139. Butterweck, V & Khan SR 2000, ‘Herbal medicines in the management of urolithiasis: alternative or complementary?, Planta Med, vol. 75, pp. 1095-1103. Davison, AM, Cameron, JS, Grunfeld, JP, Ponticella, C, Ritz, E, Wineras, CG & Ypersele, CV 2005, Oxford textbook of clinical nephrology, Oxford university press, New York. Dipiro, JT, Talbert, RL, Yec, GC, Matzke, GR, Wells, BG & Posey, LM 1997, Pharmacotherapy a pathophisiology approach, Applenton & Lange, USA. College of Pharmacy, IPS Academy 27
  • 28. 7. Glew, RH, Vanderjagt, DJ, Lockett, C, Grivetti, LE, Smith, GC, Pastuszyn, A & Millson M 1997, ‘Amino acid, fatty acid, and mineral composition of 24 indigenous plants of Burkina Faso’, Journal of Food Composition and Analysis, vol. 10, pp. 205-217. 8. Hennequin, C, Lalanne, V, Daudon, M, Lacour, B & Drueke, T 1993, ‘A new approach to studying inhibitors of calcium oxalate crystal growth’, Urol Res, vol. 21, pp. 101-108. 9. Kirubha, TSV, Nagavalli, D, Hemalatha, S & Karunambigai, K 2006, ‘Diuretic activity of the extracts of Adansonia digitata leaves’, Hamdard Medicus, vol. 49, no. 4, pp. 53-54. 10. Knoll, T, Schubert, AB, Fahlenkamp, D, Leusmann, DB, Wendt-Nordahl, G & Schubert G 2011, ‘Urolithiasis through the ages:data on more than 200,000 urinary stone analyses’, J Urol, vol. 185, no. 4, pp. 1304-11. 11. Kulaksizoglu, S, Sofikerim, M & Cevik C 2008, ‘In vitro effect of lemon and orange juices on calcium oxalate crystallization’, Int Urol Nephrol, vol. 40, pp. 589-594. College of Pharmacy, IPS Academy 28
  • 29. 12. Okada, A, Noumura, S, Higashibata, Y, Hirose, M, Gao, B, Yoshimura, M, Itoh, T, Tozawa, K & Kohri, K 2007, ‘Successful formation of calcium oxalate crystal deposition in mouse kidney by intraabdominal glyoxylate injection’, Urol Res, vol. 35, pp. 89-99. 13. Oloyede, GK, Onocha, PK, Soyinka, I, Oguntokun, O & THonda, E 2010, ‘Phytochemical screening, antimicrobial and antioxidant activies of four Nigerian medicinal plants’, Annals of Biological Research, vol. 1, no. 2, pp. 114-120. 14. Pauly, G 2001, ‘Use of an extract of the genous Adansonia, United State Patent 6,274,123 B1. 15. Pearle, MS & Nakada, SY 2009, Urolithiasis medical and surgical management, Informa healthcare, London. 16. Pragasam, V, Periandavan, K, Sumitra, K, Srinivasan, S & Varalakshmi, P 2005, ‘Oral L-arginine supplementation ameliorates urinary risk factors and kinetics modulation of Tamm-Horsfall glyprotein in experimental hyperoxaluric rats’, Clinica Chimica Acta, vol. 360, no. 1-2, pp. 141-150. 17. Schneider, HJ, Rohrborn, C & Rugendroff EW 1983, ‘A gel model for measuring crystallization inhibitor activities in calcium oxalate urolithiasis’, World Journal of Urology, vol. 1, pp. 155-158. College of Pharmacy, IPS Academy 29
  • 30. 18. Selvarani, V & James HB 2009, ‘Multiple inflammatory and antiviral activities in Adansonia digitata leaves, fruits and seeds’, Journal of Medicinal Plants Research, vol. 3, no. 8, pp. 576-582. 19. Selvarani, V & James HB 2009, ‘Multiple inflammatory and antiviral activities in Adansonia digitata leaves, fruits and seeds’, Journal of Medicinal Plants Research, vol. 3, no. 8, pp. 576-582. 20. Sidibe, M & Williams, JT 2002, ‘Baobab Adansonia digitata L.’, International centre for underutilised crops, UK. 21. Vertuani, S, Bracciol, E, Buzzoni, V & Manfredini, S 2002, ‘Antioxidant capacity of Adansonia digitata fruit pulp and leaves’, Acta Phytotherapeutica, vol. 5, no. 2, pp. 2-7. 22. Woolfe, ML, Chaplin, MF & Otcher G 1977, ‘Studies on the mucilages extracted from okra fruits (Hibiscus esculentus L.) and baobao leaves (Adansonia digitata L.)’, Journal of Science, Food and Agriculture, vol. 28, pp. 519-529. 23. Yazzie, D, Vanderjagt, DJ, Pastuszyn, A, Okolo, A & Glew, RH 1994, ‘The amino acid and mineral content of Baobab (Adansonia digitata L.) leaves’, Journal of Food Composition and Analysis, vol. 7, pp. 189-193. College of Pharmacy, IPS Academy 30
  • 31. College of Pharmacy, IPS Academy 31