Royal Jelly ◼ Wax ◼ Apitoxine (Bee Sting) ◼ Bees (Homeopathy) Bee Products – Food Or Medicines

Therapeutic properties of Moroccan bee products

Pr Badiaa LYOUSSI University Sidi Mohamed ben Abdallah Fez, Morocco

rats

Food as Medicine ?

Prevention

is better than

Cure The Primary Law of Healing ″Let thy food be thy medicine‶

Hippocrates ( 460 – 370 BC) Apitherapy – general concepts: The use of beehive products for nutrition, Quality health and quality of life Concentration improvement, prevention Dose / Time and treatment of different pathologies

SYNERGY Of the bee products Modern Apitherapy All beehive products have a medical use :

◼ Honey and Comb honey ◼ Pollen and Beebread ◼ Propolis ◼ Royal Jelly ◼ Wax ◼ Apitoxine (bee sting) ◼ Bees (homeopathy) Bee products – food or medicines

Product Nutritional value Therapeutic importance

Pollen FOOD Medicament Honey FOOD Medicament Royal jelly Food Medicament PROPOLIS None MEDICAMENT Venom None Medicament Some honeys from Morocco

Plant Origin Indication Crataegus Ifrane cardiotonic

Camomille Gharb antidepressant Siliqua (Carroube) Moyen Atlas astringent Eucalyptus Gharb antiseptic Euphorbes Béni mellal immunity Arbutus unedo Meknes diuretic, hypoglycemic Jujube Khénifra aphrodisiac Lavande Oulmès sedative Capparis spinosa Tifelt weight loss, hypoglycemic Nigelle Gharb allergies Oranger Gharb Sedative Romarin (Azir) Midelt hepatic Sauge Midelt menopause Ruta chalepensis Moyen Atlas Anti inflammatory Thym Sousse Antiseptic Bupleurum (Zandaz) Immouzzer Antidiabetic, Depurative Euphorbia resinifera Region : North Africa Country : Morocco Vernacular name : zaggûm, zaqqûm, zakkûm, banan el-ârd (Tadla), euphorbe résinifère

Name : Euphorbia resinifera Berg. Familly : Euphorbiaceae

Euphorbia resinifera contains a high concentration of the toxin resiniferatox for the development a novel class of analgesics. Recent research has shown that this toxin exhibits its effects by interacting with TRPV1, a known pain sensing cation channel that also responds to capsaicin, the primary vanilloid compound found in hot peppers Bupleurum spinosum Hayyârâ : Family : Apiaceae Genus: Bupleurum

Bupleurum spinosum (Buplèvre épineux, akerbaz) (Apiaceae) Constituents: saponins, flavonoids, coumarins Properties : Anti-inflammatory-Tonic- Sudorific-Carminative- Emmenagogue, fever, hemorrhoids protector of the liver, hepatitis B The buplèvre honey (zandaz) Arbutus unedo ( Ericaceae )

Phenols : 790 ± 12 (mg GAE/Kg)

Flavonoids : 37.9 ± 1.8 ( mg QE /Kg)

ORAC : 21.7 ( µM TE/g ) السدرة , Jujubier sauvage

Nom commun : Jujubier. Noms botaniques : Zizyphus jujuba, syn. Z vulgaris, Z. spinosa, famille des rhamnacées. Noms anglais : Black date, chinese jujube, The sedative effects of the plant jujube plum, red date. would be attributable to its content in triterpenes. One of the components of the core of its fruit, the jujuboside A, would reduce the hyperactivity of neurones. Experimental data from animals reveals that the jujubier has marked anti-inflammatory effects, which plaid The jujuboside in favor of it’s use in allergic rhinitis terpene and asthma Increases Nitric oxide

Increases Decreases Insulin in Prostaglandins diabetes

Decreases Decreases fasting triacyglycerol, blood sugar in cholesterol honey normal and and LDH diabetics

Decreases Increases CRP and homocysteine HDL Decreases Decreases Insulin in Leptin, body normal weight subjects

Increases antioxidants

Some biological activities of natural honey HONEY Some advantages… compared to sugar !

Sweetening power

 Calorie content (Kcal) for 100g Glycemic index < from 10% to 50% PHYSICOCHEMICAL CHARACTERIZATION OF HONEY

Moisture (%) Proline (mg/kg)

Ash (%) Diastase (DN)

Conductivity (mS/cm)

Sugar (g/kg) Color (mmPfund)

HMF (mg/kg) pH & Acidity (mEq NaOH/Kg)

Physicochemical results obtained for some Moroccan honeys from beekeeper associations

Sample pH Free acidity Lactone Total Moisture Conductivit Ash Proline Diastase ( mEq/Kg ) acidity acidity % y % (mg/Kg) (Shade ( mEq/Kg ) (mEg/Kg) µS/cm units/g)

Capparis 3.75  16.7  0.59 5.47  0.51 22.10.8 18.50  484.333.57 0.28  754.33  3.65  0.47 0.02 0.09 0.013 10

Carob 3.90  15.17  0.59 7.27  0.51 22.43  20.00  640.00  0.475  747.33  15.70  0.47 0.02 0.75 0.09 3.57 0.013 10

Eucalyptus 3.79  23.27  0.59 5.53  0.51 28.90  19.10  594.33  0.39  0.013 1047,33 13.46  0.47 0.02 0.75 0.09 3.57 10

Euphorbia 3.68  27.18  0.59 9.37  0.51 36.55  18.00  444.00  0.330  829,33  8.05  0.47 0.02 0.75 0.09 3.57 0.013 10

Nigella 3.73  20.92  0.59 8.03  0.51 28.95  17.90  308.67  0.210  287.67  8.36  0.47 0.02 0.75 0.09 3.57 0.013 10

Harmala 4.05  16.72  0.59 5.37  0.51 22.09 0.75 17.53  443.00  0.280  598.33  5.70  0.47 0.02 0.09 3.57 0.013 10

Thyme 3.71  29.4 0.59 8.41  0.51 37.84  18.33  639.33  0.245  524.33  12.96  0.47 EDTA

Folin-Ciocalteu

Fluorescéine Polyphénols Trichlorure d’aluminium Flavones Inhibition et AChE flavonols

Pouvoir Inhibition chélateur LOX

Méthodes Pouvoir ORAC réducteur

Acide thiobarbiturique DPPH

TBARS DPPH

HRSA ABTS

ABTS Ferricyanide de potassium Antioxidant activities of some Moroccan honeys from beekeeper associations

Sample Phenol Flavonoids TEAC ORAC Chelating NO (mg GAE/100 (mg QE/100 g) IC50 = (mg/ml) (µmol/TB/g ) (IC50 = mg/ml) (IC50 = mg/ml) g)

Capparis 691.54  14.69 71.49  1.65 16.7  0.59 5.47  0.51 22.1  0.8

Carob 573.01 14.69 126.85 1.65 7.28  0.59 13,45  0.61 - 93.05  2.85

Eucalypt 637.78  14.69 32.94  1.65 21.00  0.62 10.13  0.61 22.62  1.26 67.58  3.11 us

Euphorb 659.86  14.69 139.62  1.65 16.18  0.62 9.88  0.59 47.86  1.26 95.14  3.11 ia

Nigella 305.86  14.69 16.85  1.65 11,55 0.59 17.14  0.59 - 37.81  3.11

Harmala 890.58  14.69 47.72  1.65 11.65 0.59 9.57  0.61 37  1.26 90.81  3.11

Thyme 736.03  14.69 91.36  1.65 4.49  0.62 14.57 0.59 52.85  1.26 34.05  3.11

Zizyphus 614.96  14.69 47.54  1.65 31.00  0.62 9.60  0.61 50.02  1.26 62.31 3.11 Sodium ( mg/Kg) 450 400 350 300 250 200 150 100 50 0

mg/kg Potassium ( mg/kg) 1200 1000 800 600 400 200 0 Magnesium (mg/kg)

250

200

150

100

0 Caprier Carob Eucalyptus Euphorbia Pennyroyal Multifloral 1 Multifloral 2 Nigelle Salvia Thyme Zizyphus

Calcium ( mg/kg) 300

250

200

150

100

0 Sugar content (g/kg) in Moroccan honeys

450

400

350 Fructose

Glucose 300 Turanose 250 Maltose

200 Trehalose

Melizitose 150 Sucrose

concentration (g/kg) concentration 100

0 Capparis spinosa Carob Eucalyptus Flavonoids ( mgQE/100g) 160

140

120

100

0 Phenols ( mg GAE/100 g) 1000

900

800

700

600

500

400

300

200

100

0 45 ORAC ( µmol TE/g) 40

0 Honey Ingestion ❖Honey (1.2 g/kg body weight honey dissolved in 250 ml of water during a 2-week ) increases antioxidant agents.; blood vitamin C concentration by 47%, beta- carotene by 3%, uric acid by 12%, and glutathione reductase by 7%.

Al-Waili NS, J Med Food. 2003 Summer;6(2):135-40 Effect of ingestion of honey on some blood parameters

Variables Water Natural honey

Fasting blood glucose (mmol/l) 6.15 ± 0.79 5.12 ± 0.86 Creatinine (mmol/l) 0.096 ± 0.009 0.083 ± 0.01 Sodium (mmol/l) 142 ± 1.54 141.2 ± 3.12 Potassium (mmol/l) 4.4 ± 0.63 4.18 ± 0.26 Chloride (mmol/l) 97.8 ± 3.97 97.5 ± 1.04 Calcium (mmol/l) 2.38 ± 0.12 2.33 ± 0.06 Magnesium (mmol/l) 0.785 ± 0.06 0.791 ± 0.05 Plasma osmolality (mOsmol/l) 296.6 ± 4.42 292 ± 4.5

Honey decreased serum creatinine (14%) and fasting glucose (17%) Evaluation of healing effect of honey in mice

Physiological serum

Madecasol

Honey

Day 1 Day 7 Day 12

➢ The ratio of the wound area was calculated in the days 1, 3,7 and 12. The surface of the wound did not undergo significant changes after treatment with physiological saline, whereas treatment with honey studied causes a reduction in the wound area from the seventh day to significantly (P <0.05), and that becomes more significant (P <0.01) to the twelfth day.

➢ Treatment with the ointment madécassol which is commonly used in wound healing and is known for its beneficial effect, causes a reduction in the wound area from the seventh day significantly (P <0.05 ), which is still significant (P <0.05) to the twelfth day. Oral gavage

Gavage Sang total

Centrifugation

➢ Dosages: •Na+, K+ Cage métabolique •Glycemia •Creatinine

Urine Diuretic activity of Moroccan carob honey

15 Control Furosemide ****** Honey *** *** *** ** ** *** *** *** *** *** *** 10 *

D.U.(ml/24h) 5

D1 D2 D3 D4 D5 D6 D7 D8 D9 Days

100 150 Control Control 4 Control Furosemide *** Furosemide *** Furosemide Honey Honey Honey *** 80 *** 3 100 *** *** *** 60 *** * 2

40 K+(mmol/l)

Na+(mmol/l) 50 1 20 Clearance(ml/min)

0 0 0 D1 D9 D1 D6 D9 D1 D6 D9 Days Days Days

Carob honey has diuretic, natriuretic and kaliuretic activity without side effects of hypokalemia that was observed with use of furosemide

R. El-Haskoury , B. Lyoussi, Afr J Tradit Complement Altern Med. (2015) Influence of carob honey on the markers of oxidative stress after carbon tetrachloride intoxication in rats

150 10

8 ***a ***a 100 6 ***b

50 4

MDA (nmol/ g tissue) (nmol/ MDA PCO(µmol/mg prot) PCO(µmol/mg 0 0

Honey CCL4 Control CCL4 Control Honey Honey+CCL4 Honey+CCL4

0.15

***a 0.10 **b

0.05 AOPP(nmol/mgprot) 0.00

CCL4 Control Honey

Honey+CCL4 Effect of carob honey on the activities of catalase , glutathione peroxidase (GPx) and ascorbic acid (AA) in liver of rats treated with CCl4

10 3 8 ***b *b ***a 6 2 ***a 4 1 2

CAT(µmol H2O2/mg prot) H2O2/mg CAT(µmol 0

0 GPx (nmol GSH/min/mg prot) GSH/min/mg GPx (nmol CCL4 Control Honey CCL4 Control Honey

Honey+CCL4 Honey+CCL4

250 ***a 200 ***b

150 ***a

100

AA (µg/ g tissue) AA 50

CCL4 control Honey

Honey+CCL4 Effect of Carob honey on CCl4-induced hepatotoxicity-related parameters in rats.

Treatment Groups ALAT(U/L) ASAT(U/L) PAL(U/L) LDH(U/L) Glu(g/l)

Normal control 30,33+3,78 73,06+7,74 222,20+19,38 648,41+60,34 1,48+0,08

CCl4 55,3+6,73*** 126,35+7,25 *** 374,68+17,59*** 1143,41+54,25*** 2,05+0,13***

Honey 26,95+2,92 62,12+8,14 208,08+15,52 629,79+32,07 1,49+0,17

Honey +CCl4 35,35+6,11*** 89,95+3,83*** 292,05+17,10*** 781,16+49,68*** 1,55+0,17***

All values represent mean ± SEM. *푃 < 0.05; **푃 < 0.01; ***푃 < 0.001; ANOVA, followed by Tukey's multiple comparison test. . Renal Hypertrophy Normal

Nephrotoxic agents

K2Cr2O7 & HgCl2 Lead Lead

• A chemical with “known” toxicity • Toxic effects define “Level of Concern” Protective effect of carob honey against lead-induced anemia in rabbits

Effect of lead and honey on blood hemoglobin. (*P < 0,05 ; **P < 0,01 ; ***P < 0,001, (as compared with control). Protective effect of carob honey against lead-induced hepato-renal toxicity in rabbits Carob honey has marked protection effect against lead-induced blood, hepatic and renal toxicity. The mechanism of action might be due to modulation of prostaglandins, nitric oxide production, and antioxidant and antiinflammatory effects of honey. Further studies are required to explore the mechanism of action and whether our results have a future clinical application. Nigella sativa honey

Antidiabetic, Antihypertensive, Diuretic, Antilipemic, Antifertility Antiasthmatic, Antispasmodic Antimicrobial, Antifungal, Antihelimintic , Anticancer, Antioxidant, Immunomodulator, Analgesic, Antiinflammatory, Hepatoprotective, Renal protective, Constituents Essentiaf oil: 0.4 to 2.5 % p-cymene (38%) thymoquinone (30%) α pinene (5 to 14 %), β-pinene (5%) limonene (4%) Thymohydroquinone

Fatty oil: 30 to 35%, linoleic acid, oleic acid, palmitinic acid, stearinic acid. eicosadienoic acid,

Alkaloids: nigellidine and nigellicine, Tocopherols Sterols Triterpene saponins Anthraquinone derivatives Treatment impact by Nigella sativa honey alone or combined with essential oil of Nigella on SBP in SHR and WKY rats SHR 105 100 Rats Contrôles

95 + Rats treated with honey + + + 90 and EO 85 Rats treated with honey

* * * traitement traitement ) - 80 * * * * 75

SBP SBP WKY * : P< 0,05 versus contrôle 105 + : P< 0,05 versus contrôle 100 95 90 + *

85 ( ( valeur la de % pré du 80 75 -1 0 1 2 3 4 5 6 7 8 9 Time ( Days )

Bees have been observed scraping the protective resins of flower and leaf buds with their mandibles and then carrying them to the hive like pollens on their hind legs Les abeilles protègent la ruche avec la Propolis

Chemical Composition of propolis Biological activity of propolis extracts

❖ The most important compounds in propolis are natural flavonoids that might be modified by an enzyme existing in the honeybee saliva The most important properties attributed to flavonoids are:

❖ Antimicrobial properties (Ghisalberti, 1979) ❖ Antiiflammatory (Aspirin –like) ❖ Antioxidant ❖ Increase and protect vitamin C (ascorbic acid) in relation with the action on capillary vessels (Ravina, 1969) ❖ i.e decrease the permeability of capillaries (vitamin P action (Sczent Gyorgyi, 1936) ❖ Stabilize collagen through inhibiting the hyaluronidase ❖ Anti-haemorrhage ❖ Influence in vitro of some flavonoids on the enzymatic metabolism of mucopolysaccharides from saphenous veins (Niebes and Luszt, 1971). Kidney: a vital organ but  Drugs (antibiotics, analgesics) - Solvents (halogenated vulnerable to toxicants hydrocarbons, ethylene glycol) Nephrotoxic  Pesticides (paraquat) - Mycotoxins (ochratoxin A) - Metals (Pb, Cd, chemicals Hg, Al, Cr, U, As, etc.) Comparison to other healthy food

The IC50 values of vegetables and fruits for suppression of the AhR transformation by 1nM TCDD

vegetable or fruit IC50 (dry µg/ml) vegetable or fruit IC50 (dry µg/ml)

Brazilian Green carrotPropolis 60 2 parsley 100 leek 110 Bamboo shoot 120 spinach 180 Chinese cabbage 190 eggplant 250 Broccoli 280 cabbage 290 tomato 310

Green pepper 410 garlic 470 corn 630 blueberry 810 grape 1200 Sweet potato 1400 onion 2000 melon >2000 orange >2000 potato >2000

The IC50 values were determined from corresponding dose-dependent curve by gel retardation assay Toxicity : Mechanism of action via Ah receptor Morocco Alcoholic and ethyl acetate extracts of propolis from Morocco after analysis by high performance liquid chromatography/electrospray ionisation-mass spectrometry revealed the presence of flavonoids also present in Ceratonia siliqua, Olea europeae, which constitute the predominant flora where propolis samples were collected .

(wogonoside, quercetin- arabinoseglucoside, apigenin dihexoside, rhamnetin hexoside, baicalin or wogonin glucoside, rhamnetin or isorhamnetin, saphnin or daphnitin, afzelechin- catechin dimmer, among other flavonoids)

Hassan Ait Mouse et al., 2012

Phenol and flavonoid content in samples of propolis from diverse regions of Morocco

Sample Phenol (mg/g) Flavonoid (mg/g)

Moulay Bouslham 5.98±1.12f 1.75±1.39d Sidi ifni 6.82±1.12f 1.80±1.39d Bhalil 12.86±1.12e 3.25±1.39d Zawiat chikh 5.34±1.12fg 1.87±1.39d Rabat 53.51±1.12c 33.31±1.39a Sidi sliman 1.45±1.12gh 0.30±1.39d Khamissat 65.67±1.12b 12.78±1.39c Larache 6.00±1.12f 2.05±1.39d Kenitra 0.74±1.12i 0.20±1.39d Oujda 44.73±1.12d 34.27±1.39a Immouzzer 91.22±1.12a 26.30±1.39b Taza 7.83±1.12f 1.68±1.39d Taounat 5.89±1.12f 0.93±1.39d Sefrou 6.211±1.120f 1.05±1.39d

Results are shown as the mean ± SD (n=3). In the same column, values with the same letter are not significantly different (P<0.05).

Miguel, Lyoussi, Food Science Biotechnology, 2014 IC50 values (mg/ml) found for the extracts of propolis from diverse locations of Morocco and obtained by maceration. For ORAC method, IC50 values are given as Trolox Equivalent (TE)

1 Sample ABTS (mg/ml)1 DPPH (mg/ml)1 Hydroxyl (mg/ml) 1 ORAC mmol TE/g

Moulay Bouslham 0.279±0.016c 0.523±0.031cd 1.398±0.008b 1106.423±78.151e

Sidi ifni 0.236±0.016cd 0.384±0.031e 0.719±0.008e 1570.866±78.151c

Bhalil 0.297±0.016e 0.239±0.031f 1.111±0.008d 1404.360±78.151bcd

Zawiat chikh 0.366±0.016b 0.409±0.031e 1.441±0.008a 1198.169±78.151cde

Rabat 0.136±0.016e 0.008±0.031g 0.104±0.008k 1455.039±78.151bc

Sidi sliman 1.009±0.016a 1.125±0.031b - 1353.256±78.151abcd

Khamissat 0.0126±0.016f 0.025±0.031g 0.059±0.008l 2012.152±78.151a

Larache 0.408±0.016b 0.559±0.031c 1.191±0.008c 1278.603±78.151cde

Kenitra - 1.813±0.031a 0.540±0.008i 1291.157±78.151cde

Oujda 0.022±0.016f 0.025±0.031g 0.398±0.008j 1143.483±78.151de

Immouzzer 0.009±0.016f 0.019±0.031g 0.078±0.008l 1865.506±78.151b

Taza 0.209±0.016d 0.294±0.031f 0.577±0.008h ND

Taounat 0.297±0.016c 0.459±0.031e 0.651±0.008g ND

Sefrou 0.296±0.016c 1.086±0.031b 0.677±0.008f ND

Miguel, Lyoussi, Food Science Biotechnology, 2014 Pearson correlation coefficients among compounds/antioxidant activities,compounds/acetylcholinesterase inhibition, compounds/lipoxygenase inhibition

Phenols Flavonoids

DPPH -0.623** -0.592**

ABTS -0.609** -0.535**

Hydroxyl -0.743** -0.647**

TBARS -0.640** -0.774**

ORAC +0.676** +0.218NS

Lipoxygenase -0.752** -0.743**

Acetylcholinesterase -0.873** -0.866**

Pearson Correlation significance levels: NS: not significant. -: Assay not performed ** significant at P < 0.01.

Miguel, Lyoussi, Food Science Biotechnology, 2014

Table 2. Enzyme inhibitory activities (IC50 mg/ml) of hydro-alcoholic extracts of Moroccan propolis harvested at different places

Sample Acetylcholinesterase -Amylase -Glucosidase Lipoxygenase Tyrosinase Xanthine oxidase

1 0.009±0.051i 0.065±0.097l 0.006±0.016ef 0.020±0.019n 0.050±0.192j 0.014±0.052i

2 - 2.020±0.097f - 0.463±0.019cde 0.374±0.192ij 0.250±0.052defg

3 0.535±0.051fg 2.066±0.097ef 0.056±0.016cdef 0.406±0.019efg - 0.245±0.052defg

4 0.446±0.051g 2.730±0.097c 0.068±0.016cd 0.653±0.019a 0.971±0.192hi 0.237±0.052defg

5 0.199±0.051h 3.304±0.097b 0.103±0.016c 0.568±0.019b 3.824±0.192ab 0.279±0.052def

6 1.453±0.051c 2.417±0.097d 0.062±0.016cde 0.646±0.019a 2.444±0.192d 0.585±0.052c

7 2.034±0.051b 1.263±0.097hi - - - 0.753±0.052b

8 0.188±0.051hi 1.355±0.097hi 0.032±0.016def 0.478±0.019cd 1.595±0.192fg 0.362±0.052de

9 0.087±0.051hi 2.144±0.097def 0.027±0.016def 0.441±0.019def 1.844±0.192ef 0.245±0.052defg

10 0.007±0.051i 0.170±0.097l 0.007±0.016ef 0.086±0.019m 0.451±0.192hij 0.031±0.052i

11 1.341±0.051cd 1.249±0.097hi 0.018±0.016def 0.642±0.019a - 0.179±0.052efgh

12 1.212±0.051d 2.853±0.097c - 0.505±0.019c 4.286±0.192a 0.381±0.052d

13 0.648±0.051f 3.632±0.097a 0.746±0.016a 0.372±0.019gh - 0.162±0.052fgh

14 0.002±0.051i 0.855±0.097j 0.019±0.016def 0.239±0.019k 0.295±0.192j 0.219±0.052defg

15 0.024±0.051i 1.484±0.097gh 0.027±0.016def 0.152±0.019l 0.612±0.192hij 0.076±0.052hi

16 0.007±0.051i 1.450±0.097gh 0.025±0.016def 0.256±0.019jk 3.600±0.192bc 0.125±0.052fgh

17 0.003±0.051i 0.146±0.097l 0.014±0.016def 0.065±0.019mn 0.110±0.192j 0.017±0.052i

18 0.123±0.051hi 2.338±0.097de 0.155±0.016b 0.574±0.019b - 0.619±0.052bc

19 0.871±0.051e 0.524±0.097k 0.176±0.016b 0.324±0.019hi 2.288±0.192de 0.279±0.052def

hi k def g def 20 0.160±0.051 0.493±0.097 0.015±0.016 0.312±0.019ij 1.047±0.192 h 0.279±0.052

21 0.009±0.051i 3.471±0.097ab - 0.425±0.019defg - 3.116±0.052a

22 0.013±0.051i 1.080±0.097ij 0.038±0.016def 0.253±0.019jk 3.173±0.192c 0.252±0.052defg

23 0.013±0.051i 0.018±0.097l 0.002±0.016f 0.046±0.019mn 0.037±0.192j 0.008±0.052i

24 3.555±0.051a 1.692±0.097g - 0.393±0.019fg - 0.391±0.052d 10

6 Phenols 5 Flavones Dihydroflavonols

4 Concentration 3

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Samples

Figure 1. Concentration of total phenols [mg Ferulic acid equivalent (FAE)/ml extract]; flavonols and flavones [mg Quercetin equivalent (QE)/ml extract]; and flavanones and dihydroflavonols [mg Eriodictyol equivalent (EE)/ml extract]. 100,0

90,0

80,0

70,0

60,0

50,0

40,0 Inhibition (%) Inhibition

30,0

20,0

10,0

0,0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Samples

Figure 2. Percentage of hyaluronidase inhibition obtained from propolis extracts (33.33 mg/ml) collected at different places of Morocco. Chemical profile of US propolis samples obtained by GC–MS. Results are shown as % of total ion current Propolis:

Is there a potential for the development of new and efficient antimicrobial agents ? Bacterial response to host mimics

Propolis

Target genes

AHL mimics, QS gene expression inhibitors … There is some evidence that propolis may be able to make equivalent (but chemically different) signals that can be detected by the bacterial communication systems and in some cases can interfere with the bacterial conversations and even inhibit virulence gene expression. Propolis contains compounds that suppress QS responses AHL Propolis compounds

+ AHL Target genes Propolis constituent(s) antagonize signaling in lasR-dependent biosensors Quorum sensing and biofilm regulation in Pseudomonas aeruginosa

Razamiravaka & El Jaziri 2014 P7 have no effect on growth and on gene transcription in P. aeruginosa P7 have no effect on lasI/R system in P. aeruginosa P7 affects rhlI/R system in P. aeruginosa P7 reduces biofilm production in P. aeruginosa

Affects biofilm lifecycle/maturation

Biofilm (24 h) Biofilm (2 x 24 h) treated with P7 for 24 h P7 improves tobramycine penetration in Psedomonas aeruginosa biofilm

(a) (b)

+Tobramycin (100 µg/ml)

One-day old culture Two-day old culture +DMSO at culture initiation

+Tobramycin (100 µg/ml)

One-day old culture + P7 at culture initiation Two-day old culture Propolis: Is there a potential for the development of new and efficient antimicrobial agents ?

➢ Propolis plays a key role in the prevention and control of bacterial invasions

➢ Propolis has an inhibitory effect on the expression of virulence genes of some pathogenic bacteria without affecting bacterial growth .

❖In Psedomonas aeruginosa PAO1, propolis inhibit the expression of LasI/R and RhlI/R, two main Quorum Sensing ( QS ) regulatory mechanisms in this opportunistic bacterium.

❖ Propolis also affect the production of biofilm. This alteration of the biofilm architecture allowed a better penetration of tobramycin into the biofilm and increases the accessibility of the antibiotic to encapsulated bacteria in the extracellular matrix . Propolis aqueous extract has protective and anti-apoptotic effects in radiation- induced mucositis in rats.

➢ AEP (850 mg/kg) was effective in reducing the severity of degenerative changes observed after acute irradiation.

➢ AEP may be useful as an adjuvant during radiotherapy to reduce the tendency to develop radiation enteritis.

M. Khayyal Apoptotic markers ➢ Cytosolic calcium content ➢ Mitochondrial respiratory chain complex I activity ➢ Mitochondrial cytochrome C (pro-apoptotic protein) ➢ Mitochondrial B-cell lymphoma-2 (Bcl-2) (anti-apoptotic protein) Oxidative stress biomarkers

AEP suppresses radiation induced rise in intestinal TBARS and depletion in intestinal GSH SHR rats

Honey

Honey + propolis Fig. 2. Sub-chronic diuretic effect: diuretic activities of daily oral doses (1g/kg BW) of Capparis honey, propolis ( 100 mg/kg BW) alone or combined honey and furosemide (10 mg/kg BW) administered for 21 days. The volume of 24 h urine was measured on Days 1 through 21 of treatment and reported as Mean ± SEM, for seven rats in each group *P<0.05 Compared to Control, **P<0.01 Compared to Control, **P<0.001 Compared to Control (ANOVA Followed by Tukey's Test). Sub-chronic natriuretic and kaliuretic activity: effect of daily oral doses (1g/kg BW) of honey , 100 mg/Kg of propolis extract alone and combined, furosemide (10 mg/kg BW) administered for 21 days on urinary excretion of Na+. The reported Na+ levels (mean±S.E.M.) are in pooled 24 h urine for each day for each group of seven rats each group. *P< 0.05; **P< 0.01; ***P< 0.001 compared with controls using Student’st-test

Effect of Sub-chronic oral administration of Capparis honey, propolis alone or combined and furosemide of creatinine clearance in normal rats. .

Diabetes mellitus promotes The anti-oxidants present overproduction of free radicals in the body offer protection - reactive oxygen species - against reactive oxygen superoxide anion – species which act as – oxidants – these cause diabetes-induced pathologies

Antioxidants

If the oxidants are more than the anti-oxidants, the body faces oxidative stress OXIDATIVE STRESS MDA, GSH levels and antioxidant enzymes activities (SOD, CAT) in the pancreas tissue of adult rats : Controls and Diabetic Groups)

C C + EPE STZ STZ + EPE

MDA 37.2 ± 1.9 45.6 ± 2.5 100.03 ± 10.7* 60.9 ± 4.2* ( nmol/g tissue)

GSH 111.8 ± 1.6 137.81 ± 10.1* 91.97 ± 1.29* 130.7 ± 1.9* (mg/g tissue)

SOD 82.6 ± 4.4 81.76 ± 5.10 13.7 ± 2.3* 72.2 ± 8.3 (U/mg prot)

CAT 52.6 ± 1.4 53.06 ± 2.82 20.3 ± 2.3* 36.2 ± 4.1* (µmol/min/mg prot)

EPE alleviates streptozotocin –induced oxidative stress and cell damage in rat pancreas Histological section of pancreas in adult rats A : Control, B: Diabetic, C: Diabetic + EPE, D: Control + EPE Antidiabetic activity of Moroccan propolis When given orally at a dose of 10 mg/100 g BW, to streptozotocin (STZ)-induced type 1 diabetic rats, it significantly decreased glycemia and plasma insulin

C EPE1 EPE2 GLB Antidiabetic & Hypotensive effects of EPE in Meriones Shawi rats : animal model of diabetes type 2

Without L-NOArg 180 Diabetic 160 Diabetic treated Control Diabetic Control 20 140 Diabetic treated ** Control treated * Diabetic (Amlo+Gli) (mmHg) 120 * ** 10 *

100 Systolic Blood Pressure

80 Contraction (mN/mg) 0

0 1 2 3 10 -9 10 -8 10 -7 10 -6 10 -5 Time (weeks) Noradrenaline [M]

300 Diabetic Diabetic treated 200 * Control * Control treated * ** Diabetic (Amlo+Gli)

(mg/dl) *

100 ** Serum glucose

0 1 2 3 Time (weeks) Effects of Polyphenols on Blood Vessels

Polyphenols

NO, EDHF Ang II

FADPH

Vasodilation Vasoconstriction Vasoconstriction Platelet aggregation Pro-inflammatory responses

Endothelial dysfunction Cats also eat the honey & propolis in a palatable critic animal concentrate diet / 2012 have 11 years old. Bee –Milk Beehive Caviar

Royal jelly Pharmacological activity of royal jelly

1/Antibacterial activity: royalisin against gram-positive bacteria; 10-hydroxy-trans- decanoic acid against Streptococcus aureus and Escherichia coli

2) Anti-oxidant activity: in vitro inhibition of lipid peroxidation, and protection against oxidative stress-induced injury in animals

3) Antitumor activity: against mouse leukemia, chemically-induced cervical/uterine carcinoma, breast cancer MCF-7 cells, and antiangiogenesis activity

4) Estrogenic activity: in vitro, in tissue culture and in vivo in in rats and ewes

5) Antihypertensive/vasorelaxant activity: peptides in royal jelly have angiotensin 1– converting enzyme inhibitory activity; trans-2-octenoic acid and 10-hydroxy-trans- decanoic acid decrease blood pressure in spontanously hypertensive rats; royal jelly has vasorelaxant activity

6) Immunoregulatory activity: shown in animal models Pharmacological activity of royal jelly

7) Cardioprotective effect: several studies in animals show improvement in lipid profile

8) Hepatoprotective effect: protection against the effects of hepatotoxins in rats

9) Analgesic effect: several studies

10) Antipyretic effect: several studies

11) Neuroprotective activity: stimulation of production of glial cell line-derived neurotrophic factor; 10-hydroxy-trans-2-decanoic acid increased the generation of neurons from neural stem (progenitor) cells in vitro

12) Anti-aging effect: prolonging the life span of C3H/HeJ mice

13) Hypoglycemic effect: insulin-like effect; also decrease in insulin resistance Experimental studies with royal jelly Diabetes-Hyperglycemia-Glucose Intolerance

Control

RJ Experimental studies with royal jelly Diabetes-Hyperglycemia-Glucose Intolerance

Control

RJ :100 mg/Kg BW

RJ :200 mg/Kg BW Pharmacological activity of royal jelly

Dyslipidemia Hyperlipidemia

❖Administration of royal jelly (10 g/day for ❖ In 7 healthy volunteers, given 6 g of 14 days) increased high density lipoprotein royal jelly per day for 4 weeks, serum (HDL)-cholesterol in elderly patients total cholesterol, LDL-cholesterol and (Münstedt K et al., J Alternat Complement Med very-low-density lipoprotein fraction 2009;15:329) decreased significantly (p<0.05) compared with controls (n = 7); there ❖Meta-analysis of the controlled human trials were no changes in serum HDL- of royal jelly (50-100 mg/day) to reduce cholesterol or triglycerides [Guo H et al., J hyperlipidemia showed a significant reduction Nutr Sci Vitaminol (Tokyo). 2007;53:345] in total serum lipids (10%) and cholesterol ❖Addition of royal jelly to the treatment levels (14%) and normalization of (HDL)- regimen in elderly arteriosclerotic cholesterol and low density lipoprotein (LDL)- patients decreased cholesterol and total cholesterol as determined from decrease in the lipids ratio: β/α lipoproteins (Madar J et al., Zeitschrift (Vittek J. Experientia 1995;51:927) Alternsforsch1965;18:103) Clinical studies with royal jelly Diabetes-Hyperglycemia-Glucose Intolerance

In 20 female type 2 diabetic patients, 10 were administered orally 1000 mg/day royal jelly and 20 were given placebo for 8 weeks

❖Mean fasting glucose decreased significantly (from 163.1 + 42.5 mg/dl to 149.7 + 42.7 mg/dl)

❖HbA1C decreased (from 8.7 + 2.2 to 7.05 + 1.45%. P = 0.001)

❖Serum insulin increased (from 70.3 + 29.2 pmol/l to 86.5 + 27.5 pmol/l, P = 0.01)

These results suggest that royal jelly supplementation may be beneficial in controlling diabetes outcomes Pollen

100 grams of pollen

500 g of beef or 7 eggs Bee pollen stocked in beehive: a food source

More than 30 million years ago bees have developed a method of food preservation that is richer in protein than meat or fish at a temperature of 36°C in an extremely humid atmosphere.

The role of fermenting agents in bee pollen

But bee pollen is also a excellent food for humans… 14:58 Floral origin – bioactivity compounds

• Anaemia and the hemopoietic system – bee pollen - Iron

• Antioxidant activity – Eucalytus globulus – flavonoids + ??

• Anti inflammatory activity - Pinus densiflora – flavonoids ??

• Antitumoral activity – Brassica napus – polysaccharide LBPP

• Antiartherosclerotic activity - fat-soluble pollen extract

• Activity on osteoporosis – Cistus ladaniferus – PM < 1000

• Activity in Disorders of the Prostate – Hidroxamic acid

• Antidiarrhoeal activity - Eucalytus globulus; Salix atrocinera

• Antimicrobial activity – Ranunculus sp – herbacetin (flavonoid) Nutritional quality of fresh monofloral bee pollen

✓Energy intake from simple and complex carbohydrates

✓Protein Source

✓Fiber Source

✓ Special Lactic bacteria source with « probiotic effect »

✓Vitamins and Minerals source

✓Vegetal antioxydant source (carotenoids, polyphenols) Bee pollen is a good food for human nutrition with many potential effect on Health Pollen and Heath claims

In total : • 7 minerals for Action on : Zinc, Manganese, Copper, Potassium, Calcium, Iron, Magnesium • 14 fundamental physiological functions based on • 35 health claims officially licensed - proven and recognized European and international authorities This shows pluripotency action pollen flowers all dehydrated. Natural action (term regulated by the EEC directive use) Pollen and health claims 14 fundamental physiological functions based on

Cardiovascular System: coagulation calcium oxygen transport: iron blood pressure: potassium acid-base balance: zinc iron transport: copper fluid and electrolyte balance: magnesium synthesis of hemoglobin and red blood cell: iron

Immune system: copper, zinc and iron Energy metabolism: copper, manganese, magnesium, iron and calcium Cellular functions: DNA: zinc proteins: zinc and magnesium amino acids zinc cell division and specialization: zinc, magnesium, iron and calcium Oxidative stress: zinc, copper and manganese Antioxidant capacity of pollen Bee POLLEN

B HO O

A C OH OH O

Free radical scavenger activity Pollen gives a Phenolic and polyphenolic profile as a fingerprint of each specie

Campos M. G., Webby R. F., Markham K. R., 2002. The Unique Occurrence of the Flavone Aglycone Tricetin in Myrtaceae Pollen. Z. Naturforsch. 57c, 944-946 Markham K., Mitchel K., Campos M. G., 1997. An unusually lipophilic flavonol glycoside from Ranunculus sardous Crantz. pollen - Phytochemistry, 45 (1), 203-204. Markham K., Campos M.G., 1996. 7- and 8-O-methylherbacetin-3-O-sophoroside from bee pollens and some struture/activity observations. Phytochemistry, 43 (4) 762-767. M.G. Campos Quercetin-3,6(?),7-O-trisubstituted Malva sylvestris pollen

Collected: 01 May 2013, Morocco

1 R2 O- R1 R3 ?

Quercetin-3,6(?),7-O-trisubstituted 2 1

R5 3 O- R4 2 R6 ?

Herbacetin-3-O-substituted

3 O- R7 Opuntia-ficus-indica pollen 1

Collected: 30 July 2013, Morocco R1 Isorhamnetin-3-O-R1

4 2 3 2 1 R2 Kaempferol-3-O-R2

R3 Isorhamnetin-3-O-R3

R4 Kaempferol-3-O-R4 Justicia adhatoda pollen 1 Collected: 30 April 2014, Morocco Cafeic acid

7 2 Quercetin-3-O-R1 5 4 6 1 2

Quercétin 3 isorhamnetin-3-o-glucoside Papaver rhoeas pollen

Collected: 21 April 2013, Morocco 3 apigenin

4 6 2 5 1 7 4 Kaempferol-3-OR1

5 luteolin Antioxidant capacity

Malva Opuntia ficus- Justicia Papaver sylvestris indica adhatoda rhoeas

Total antioxidant activity 17.84 ±1.03 39.29 ±1.88 83.03 ±5.15 58.48 ± 2.85 (mg Eq AA/g )

Total Phenolic (mg PCE/g) 9.20 ± 0.12 9.22 ± 0.05 54.05 ± 0.96 34.84 ± 0.69

Total Flavonoid (mg QE/g) 2.5 ± 0.025 2.31 ± 0.01 2.69 ± 0.04 12.95 ± 1.29 Pollen and erythrocytes

Control Anemic

Sport o Effect of pollen on blood platelets

Control Anemic Sport

Time (months) POSOLOGIES

2 8 15 60-65 years 20g 5g 15g

If denutrition whatever the cause

20-25g + 20-25g / jour

Practice Note: Do not hesitate to mix the pollen in compote or yogurt (2 doses / day at different meals)

Acute toxicity

Dose i.p After 14 D

1 mg/kg

2 mg/kg

3 mg/kg

4 mg/kg

5 mg/kg

6 mg/kg 125

100

50 Mortality (%) Mortality

DL50 = 4.15  0.33 mg/kg 0

0 1 2 3 4 5 6 7 8 Dose (mg/kg) Therapeutic Ratio

(75 Kg )

Toxic dose (LD 50 ) = ~ 210 mgr.

20 stings: ~ 3 mgr. session

200 stings: ~ 30 mgr.

Safety Ratio : between 7 and 70 CAPE Mediterranean Diet Pyramid Acknowledgments

Pr Maria G. Miguel ( University of Algarve, Faro, Portugal) Pr Mondher Jaziri ( ULB, Brussels, Belgium) Pr Maria Campos ( University of Coimbra, Portugal) Pr Bankova Vassya ( Bulgarian Academy of Sciences)

Smail Aazza ( Laboratory PPSE, USMBA, Fez) Soukaina El Gendouz ( Laboratory PPSE, USMBA, Fez) Nawal Lemnai ( Laboratory PPSE, USMBA, Fez, Morocco) Meryem Bakour ( Laboratory PPSE, USMBA, Fez, Morocco) Redouan Haskoury ( Laboratory PPSE, USMBA, Fez, Morocco) MELLI - DYNAMICS GROUP