2. Neuropharmacological & analgesic properties of Ajwah, Safawi and Sukkari Omer A. A. Hamdi*, Bassem Y. Sheikh 2 Jamil A Shilpi 3 1 Head of Alneelain Centre For natural products research and drug discovery, 1 Department of Chemistry, Faculty of Science and Technology. Alneelain University, Khartoum,Sudan. 2 Department of Surgery, College of Medicine, Taibah University, Saudi Arabia 3 Pharmacy Discipline, Khulna University, Bangladesh

3. Acknowledgements Al-Moalim MA Bin Ladin (MABL) chair for Scientific Miracles of Prophetic Medicine, College of Medicine, Taibah University (Research grant no. MABL 37/02 )

4. Facts: Date palm Fruits of Phoenix dactylifera L. (Arecaceae) Origin: Middle East Staple food in Middle East region Due to its nutritional value, it was naturalised in different parts of the world. Current status: More than 2000 cultivars grow around the world

5. Narration of date palm in Al Quran and Al Hadith “And from the fruits of date-palms and grapes, you derive strong drink and a goodly provision. Verily, therein is indeed a sign for people who have wisdom” (Surat An‐Nahl: 67). “He who eats seven dates of Madina (Ajwah dates) every morning, will not be affected by poison and magic on the day he eats them” (Sahih Al-Bukhari).

6. Ethnobotanical uses Liver disorders Diabetes Constipation, diarrhoea To reduce wrinkling of the skin To relieve asthma To alleviate headache Expectorant Ameliorating in cough, bronchitis, respiratory disorders Increase immunity Aphrodisiac, to treat sexual debility

7. Reported biological activity Antioxidant, antimutagenic Antihaemolytic Antiviral Antifungal Anti-inflammatory Antihyperlipidemic Hepatoprotective Nephroprotective Gastroprotective Anticancer Immunostimulating Gonadotropic

8. Chemical constituents Simple phenolics – p-hydroxy benzoic acid, protocatechuic acid, gallic acid, vanillic acid, syringic acid Phenylpropanoids – cinnamic acid, caffeic acid, o-caffeoyl shikimic acid, ferulic acid, sinapic acid, o-coumaric acid, p-coumaric acid Carotenoids – β-carotene, lutein Sterols – cholesterol, campesterol, stigmasterol, β-sitosterol, isofucosterol Flavonoids – catechin, epi-catechin, quercetin, luteolin, apigenin Procyaninidins and anthocyanins

9. Background of present investigation In Ayurveda date palm is known as Kharjura and is indicated for the treatment of – psychosis, anxiety, cognitive dysfunction – many of the nervous system disorders The fruit is also used alone or in combination to treat – sciatica, headache, hemicranias – applied externally for inflammatory conditions including abscess, boils and ulcers

10. Background of present investigation Literature survey on date palm revealed that some Chinese and Japanese patented herbal preparations containing date palm as one of the component is indicated for – treating sleeping disorders

11. Present investigation All these observations prompted us, as a part of our research on Prophetic medicine, to evaluate and compare – neuropharmacological and antinociceptive effects of – Ajwah, Safawi and Sukkari

12. Ajwah Unlike other dates, Ajwah dates are relatively smaller in size. Ajwah is round shaped, soft, dark brown coloured date which looks almost black with fine texture and white wrinkles. Ajwah has special interest to Muslims as it has been mentioned in the Prophetic medicine.

13. Safawi Safawi is another popular date cultivar growing in Almadinah Almunawarah, Saudi Arabia. Safawy is oval shaped soft, moist variety of dates with dark brown texture.

14. Sukkari Sukkari is the best-selling date in Saudi Arabia. These golden-brown dates have patches of lighter colour and are medium or small cone shaped with a firm exterior. This date is characteristically sweet as compared to other cultivars with chewy flesh. It grows mainly in Qassim, Saudi Arabia.

15. Collection and extraction The dried ripe (in tamar stage) dates were purchased from local date market in Al Madinah Al Munawarah, KSA. The dates were identified by taxonomists at at Taibah University. The dried dates were mashed with the help of mortar and pestle, soaked in ethanol for 3 days with periodic sonication. The extracts were filtered and dried using a rotary vacuum evaporator at 45°C. The extracts were further freeze dried to get the crude extract.

16. Test animal Young Swiss Albino mice – 4-5 weeks old and weighing 20-25 g Purchased from the Animal Resources Branch of ICCDR,B Bangladesh Acclimatisation with the laboratory condition – Temperature 25±2°C – Relative humidity: 56-60% – 12 h dark-light cycle

17. Pentobarbitone-induced sleeping time test Test mice orally treated with date extracts After 30 min, pentobarbitone (50 mg/kg, i.p.) was administered to induce sleep Time to onset for sleep recorded Duration of sleep recorded What to observe: CNS active compound will ↑ or ↓ time for onset of sleep ↑ or ↓ duration of sleep

18. Effect on pentobarbitone-induced sleeping time Treatment (n= 5) Dose (mg/kg) R/AOnset of sleep (min) Duration of sleep (min) Control10 ml/kgp.o.9.6±0.5574.0±2.0 Diazepam5i.p.3.6±0.34 d 140±2.2 f Ajwah250p.o.7.8±0.36 cd 90±2.4 ce 500p.o.6.3±0.35 ad 110±2.2 cf Safawy250p.o.8.1±0.39 c 86±2.3 cd 500p.o.7.0±0.35 cd 100±3.0 cf Sukkari250p.o.8.6±0.34 c 83±1.6 500p.o.7.4±0.24 cd 97±2.6 ce a p<0.05 vs D, b p<0.01 vs D, c p<0.001 vs D, d p<0.05 vs C, e p<0.01 vs C, f p<0.001 vs C; C=control, D=diazepam. Results: The date extracts↓ time for onset of sleep and ↑ duration of sleep i.e., showed CNS relaxing effect

19. Open field test: test for locomotor activity Test mice orally treated with date extracts Placed on a floor divided in squares Number of squares visited recorded for 3 min (observation period 3 h) What to observe: CNS active compound will ↑ or ↓ number of squares visited

20. Results of open field test Treatment (n= 5) Dose (mg/kg) Number of movement 0 min30 min60 min90 min120 min180 min Control10 ml/kg133±2123±3113±2104±2106±595±2 Diazepam5126±339±1 f 30±1 f 28±3 f 29±1 f 27±1 d Ajwah250128±289±2 cf 74±3 cf 73±2 ce 75±3 cf 81±3 cd 500129±284±1 cf 71±2 cf 67±1 cf 71±1 cf 74±2 cf Safawy250139±291±3 ce 78±3 cf 76±4 cf 79±3 ce 84±4 cd 500133±385±1 cf 73±2 ce 70±2 cf 75±2 cf 80±4 cf Sukkari250129±493±2 cf 82±3 cf 75±3 ce 73±3 cf 82±2 ce 500137±386±3 cf 77±3 cf 72±3 cf 69±3 cf 79±2 ce a p<0.05 vs D, b p<0.01 vs D, c p<0.001 vs D, d p<0.05 vs C, e p<0.01 vs C, f p<0.001 vs C; C=control, D=diazepam. Inference: The date extracts reduced the locomotor activity in test mice

21. Hole board test: test for exploratory behaviour Test mice orally treated with date extracts placed on a board having 16 evenly placed holes Head dipping through the holes recorded for 2 min (observation period 3 h) What to observe: CNS active compound will ↑ or ↓ number of head dipping

22. Results of hole board test Treatment (n= 5) Dose (mg/kg) Number of head dipping 0 min30 min60 min90 min120 min180 min Control 10 ml/kg 19±0.921±1.327±1.429±1.631±1.433±1.3 Diazepam520±1.011±0.9 f 6±1.0 f 6±0.8 f 6±0.6 f 7±0.5 f Ajwah25020±0.817±1.1 cd 14±0.8 cf 17±0.7 ce 24±0.7 ce 50020±1.216±1.2 be 12±0.9 cf 12±0.8 cf 14±1.0 cf 20±0.9 cf Safawy25020±1.017±0.9 cd 18±0.7 cf 16±0.5 cf 19±0.9 ce 23±1.0 cd 50019±0.817±1.0 ce 15±0.9 cf 13±1.0 cf 16±1.1 cf 20±0.9 cf Sukkari25020±1.018±0.7 cd 18±0.8 ce 17±0.7 cf 20±0.8 cf 23±0.9 cd 50020±0.917±1.0 cd 15±0.9 cf 15±0.7 cf 17±1.1 cf 21±1.0 ce a p<0.05 vs D, b p<0.01 vs D, c p<0.001 vs D, d p<0.05 vs C, e p<0.01 vs C, f p<0.001 vs C; C=control, D=diazepam. Inference: The date extracts reduced the exploratory behaviour in test mice

23. Acetic acid induced writhing: test for analgesic activity Intraperitoneal administration of 0.7% acetic acid causes writhing (a sign of pain) in mice Number of writhing is counted What to observe: Analgesic compound will ↓ number of writhing Peripheral mechanism of pain !

24. Effects on acetic acid induced writhing Treatment (n= 5) Dose (mg/kg) Number of writhing Control10 ml/kg33.0±1.0 Diclofenac sodium259.4±0.5 d Ajwah25023.0±0.4 cd 50021.0±0.6 cd Safawy25024.0±0.4 cd 50022.0±0.5 cd Sukkari25025.0±0.7 cd 50023.0±0.6 cd a p<0.05 vs DS, b p<0.01 vs DS, c p<0.001 vs DS, d p<0.001 vs C; C=control, DS=diclofenac sodium. Inference: The date extracts reduced peripherally mediated pain sensation in test mice induced by acetic acid

25. Hot plate test: test for analgesic activity Mice placed on hot plate maintained at the temperature of 55±0.5 o C Paw licking or jumping is a sign of pain caused by heat What to observe: Analgesic compound will ↑ response time Central mechanism of pain !

26. Results of hot plate test Treatment (n= 5) Dose (mg/kg) Response time (sec) 0 min30 min60 min90 min120 min Control10 ml/kg4.6±0.134.5±0.264.5±0.184.2±0.324.4±0.15 Morphine54.7±0.158.9±0.16 f 11.4±0.40 f 11.0±0.36 f 8.7±0.20 f Ajwah2504.3±0.105.7±0.24 cf 5.9±0.14 cf 5.0±0.10 ce 4.4±0.15 c 5004.3±0.15.9±0.27 cf 7.0±0.19 cf 6.6±0.20 cf 5.2±0.10 cd Safawy2504.6±0.155.7±0.17 cf 6.0±0.13 cf 5.3±0.19 cf 4.3±0.14 c 5004.6±0.156.6±0.20 cf 7.3±0.14 cf 6.5±0.21 cf 4.5±0.20 c Sukkari2504.2±0.125.2±0.12 c 5.6±0.15 cf 4.9±0.23 c 4.3±0.17 c 5004.4±0.145.9±0.17 cf 6.9±0.15 cf 5.8±0.12 cf 4.5±0.17 cf a p<0.05 vs M, b p<0.01 vs M, c p<0.001 vs M, d p<0.05 vs C, e p<0.01 vs C, f p<0.001 vs C; C=control, M=morphine. Inference: The date extracts reduced centrally mediated pain sensation in test mice

27. HPLC analysis for polyphenolic constituents Detection for major bioactive polyphenolic constituents in date extracts by- DionexUltiMate 3000 Rapid Separation LC system equipped with – quaternary rapid separation pump (LPG-3400RS) – acclaim ® C 18 column (4.6 × 250 mm; 5µm, Dionex USA) – in a temperature-controlled column compartment (TCC- 3000) maintained at 30°C – and photodiode array detector (DAD-3000RS)

28. HPLC chromatogram of a standard mixture of polyphenolic compounds Peaks 1: arbutin; 2: gallic acid; 3: hydroquinone; 4: (+)-catechin; 5: vanillic acid; 6: caffeic acid; 7: syringic acid; 8: (–)-epicatechin; 9: vanillin; 10: p-coumaric acid; 11: trans-ferulic acid; 12: rutin; 13: ellagic acid; 14: benzoic acid; 15: rosmarinic acid; 16: myricetin; 17: quercetin; 18: trans-cinnamic acid; 19: kaempferol.

29. HPLC chromatogram of Ajwah extract Peaks 1: (+)-catechin; 2: (-)-epicatechin; 3: trans-ferulic acid; 4: rosmarinic acid

30. HPLC chromatogram of Safawy extract Peaks 1: (+)-catechin; 2: (–)-epicatechin; 3: trans-ferulic acid

31. HPLC chromatogram of Sukkari extract Peaks 1: caffeic acid; 2: p-coumaric acid; 3: trans-ferulic acid

32. Contents of polyphenolic compounds in date extracts Polyphenolic compound Content in mg/100 g of dry extract*(% RSD) AjwahSafawiSukkari trans-Ferulic acid11.70 (0.18)5.01 (0.06)2.28 (0.06) (+)-Catechin14.67 (0.29)42.25 (0.57)- (–)-Epicatechin9.15 (0.11)21.93 (0.34)- Rosmarinic acid3.73 (0.04)-- Caffeic acid--3.11 (0.09) p-Coumaric acid--1.37 (0.05) * n=5; RSD: Relative standard deviation.

33. Conclusion Ajwah, Safawy and Sukkari dates have some degree of relaxing effect on the brain. Reduced locomotor activity, and exploratory behaviour in test mice suggest reduced CNS activity in treated mice. The extracts showed both centrally and peripherally acting analgesic activity. In all cases, the effect was milder as compared to positive control, indicating a moderate level of activity. Thus dates could be useful in producing mild relaxing effect on the brain. The effects were similar with all the three date cultivars, but relatively stronger with Ajwah dates.