High added value applications of Neem (Azadirachta indica ...neem.tea-nifty.com/neem/files/9.Dr.S. Andrew Van Der Esch.pdfPiodermitis Fistulae Rectum Prolapse Myiasis Bed sore or break

High added value applications of Neem (Azadirachta indica (A.

Juss)) derived products.

S.A. van der Esch and F. Carnevali

ENEA, Italian National Agency for New Technologies, Energy and the Environment

Hurdles to Neem development• market share of this botanical insecticide

remains very low• regulatory approval remains the most

formidable barrier to the commercialization of new botanical insecticides

• multimillion dollar regulatory costs• concentrate R&D on higher added value

applications which could justify the very high capital costs necessary to obtain regulatory approval

During our experiments of lousecontrol we had also to solve the problem due to small woundsfollowing shearing of the animalswhich very often were colonised bymyiasigenic flies. We wanted tocreate a repellent and disinfectantwhich wouldn’t interfere with the wound healing process.

Context of the research

Therapy of external wounds with MIX 557

Using our knowledge of natural substances weformulated a mix whichshould have repellenteffects against insects and which would also havebiocidal and anti-microbialeffects allowing the woundhealing process to occurwithout using hystiolesivesubstances (disinfectants, antibiotics etc…).We selected both Neem and St John Wort as the sourceplants of the necessarysubstances.


We adopted an experimentalprotocol called “Compassionate regime”

Experimental setup

It’s an experimentalapproach adopted fromoncological protocolsemployed on terminal patients

We have adapted it for patients withvery severe external wounds(traumatic, chronic and/or withcomplications) for which no alternative existed.



We thought of obtaining a repellent in order to avoid Myiasis, in the end we obtained a mixture which demonstrated to have the following properties.

Anti-inflammatoryLenitiveAnti-dehydrating

Larvae of dipteraeMicro-organisms

Myiasegenic dipteraeNon Myiasigenic dipteraeInsects

24 h

8 h

Healing

Repellent

Biocidal

MIX 557


Follows a precise sequence of phases which are clinically well defined

Wound healing

3-5 days 15 days More than a year


Wound healing

3-5 days15 dyas

More than a year


Inflammatoryphase

Proliferative phase Healing phase


Animal species and wound types treated under compassionate regime with MIX 557

Species

Traumatic wounds

Surgical wounds

Keloids

Abscess

Piodermitis

Fistulae

Rectum Prolapse

Myiasis

Bed sore or break of withers

TOT

Infected Not infected

Success Failed

Alpaca 18 18 Bovine 2 2 Dog 4 3 1 1 3 2 3 2 19 Donkey 1 1 Goat 5 5 Horse 8 8 1 9 2 4 2 34 Pocupine 1 1 Sheep 21 21

Pig 6 6

TOT 32 13 2 9 2 9 1 3 2 30 3 107


Traumatic wounds (Dog)

Day 0Day 4

Day 13Day 13

4 months4 months

Wound area = 588.99 cm2

Wound perimeter = 56.50 cmLinear healing of the wound edge per dayexpressed as cm/day

= 0.2045 cm/day


Traumatic wounds (Horse)Day 0Day 0 8 months8 months

0

50

100

150

200

250

300

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Weeks of treatment

Squa

re c

m

0

10

20

30

40

50

60

70

80

90

cm

PerimeterArea

Wound area = 450 cm2

Wound perimeter = 84 cmLinear healing of the wound edgeexpressed as cm/day

= 0.0322 cm/day


Traumatic wounds (Horse)

MIX 557MIX 557

traumatic wounds at bothcarpus regions On left screen side: MIX 557 therapyOn the right screen side: Allophathic therapy

0,00

0,01

0,02

0,03

0,04

0,05

drago dx drago sin

Healing rate: MIX557 treatment (dx) vs allopathic treatment (sin)

W. area: Right = 17.25 cm2 Left = 13.22 cm2W. perimeter: Reight = 18.06 cm Left = 14.75Linear healing of the wound edge/dayexpressed as cm/day:Right = 0.0345 Left = 0.0164


Myiasis (Sheep)

Day 0 aDay 0 a

Day 13Day 13 Day 29Day 29

Case n° 39: Time course for healing of headwound, infested with Wolphartiamagnifica maggots, in ram. Woundingoccured 1 month before onset of MIX 557 treatment. Application = once daily. No othertherapies applied. During time course no new maggots were deposited because of repellenteffect of the MIX 557. N° similar cases treated= 30

Day 33Day 33


Repellence to myiasigenic (and non) flies in vivo

Fig. 3.2–Calliphora vicina Robineau-Desvoidy (Diptera, Calliphoridae: a) pupae; b) adult; c) pupario; d) head of adult; e) stigma

Calliphora vicina

Line of treatment

After 2 hours

After 6 hours

MIX 557 Repellent effect

After 8 hours


Biocidal effect of Neem derived products on Caliphora sp.

In vitro

01020304050607080

% so

prav

vive

nza

EtOH 0,2%

olio neem 1

olio neem10

olio neem100

%pupe % adulti

Survival of pupae and adults (%) of Calliphora sp. After treatment with differentconcentrations of Neem derived products

Therapy of external wounds with MIX 557 In vitroBiocidal effect MIX 557

0

10

20

30

40

50

60

70

ControlAcqua

HO100ppm

Mix557 NO100ppm

% la

rve

vive

Survival of Calliphora sp. larvae fed with substrate containing the differentcomponents present in MIX 557.

Therapy of external wounds with MIX 557 In vitro

Ovipositioning: Repellence MIX557 mechanical

0

10

20

30

40

50

60

70

80

90

Control HO HO ControlMix

Mix Control NO NO

N°

med

io u

ova

depo

ste

Average number of eggs deposited by female Calliphora spp on natural substrate completelycovered with the different components contained in MIX 557.


Antimicrobial activityIn vitro versus

Escherichia coliPseudomonas aeruginosaStaphylococcus aureusCandida albicans

Therapy of external wounds with MIX 557 In vitroEvaluation of antimicrobial activity

Log reduction Criteria 48 hours 7 days 14 days 28 days

Bacteria A 2 3 - NI*

B - - 3 NI*

Fungi A - - 2 NI* B - - 1 NI*

Anti-microbial activity was evaluated in terms of the log reduction in the number of CFU/ml against the value obtained at zero time in the “Vitality Control” (inoculum in PBS). Criteria of acceptance prescribed by European Pharmacopoeia (2005) for Topical Preparations are given below:

The A Criteria express the recommended efficacy to be achieved. In justified cases where A Criteria cannot be attained, for example for reasons of an increased risk of adverse reactions, the B Criteria must be satisfied

NI*= No Increase with respect to 14 days


5.72.75.72.75.72.74.00.0-0.2-0.4Staphylococcus aureus (ATCC 6538)

5.12.14.10.64.11.1-0.4-0.30.5-0.9Candida albicans(ATCC 1023)

5.33.05.35.35.31.30.0-0.6-2.0-2.0Pseudomonas aeruginosa(ATCC 9027)

4.74.74.72.44.74.7-0.80.8-2.3-2.3Escherichia coli(ATCC 8739)

7d48 h7d48 h7d48 h7d48 h7d48 h

MIX 557NOHOOlive oilPBSStrain

Confrontation of Log Reduction (LR) obtained with MIX 557 and the different constituents.To be considered effective at 48 h LR = 2 and at 7 d LR = 3 for the bacteria (Criteria A). Yeast LR = 2 a 14 g (Criteria A)


Affected (skin) layers Example

Strong exsudation

Intermediate exsudation

Weak exsudation Dry Infect

Only epidermis Scrape, burns etc.

Lokal antiinfectious therapy

Down to and including subcutis

Ablederung, burns etc.

Silver products

Down to and including fascia/muscle

Trauma, comparte-ment syndrom, fasziitis etc.

Silver products, systemic antibiotics

Involvement of bone and/or organs

Polytrauma, gunshot, Platzbauch etc

Systemic antibiotics

Hydrokolloids FilmAlginate, Hydrofibre,

Polyacrylate

Alginate, Hydrofibre, Polyacrylate

Hydrokolloids Film

Vakuumtherapy


Vakuumtherapy


MIX 557

MIX 557

MIX 557

Acute wounds

Therapy of external wounds with MIX 557 Chronic woundsType of chronic

wound Prehealing-Phase

(TIME) Phase I

(Inflammatory phase)

Phase II (Proliferate phase)

(TIME)

Phase III (Maturation

& remodelling

phase)

(TIM

E)

Necr

otic

(dea

d tis

sue)

Necr

otic

& fi

brin

Necr

otic

, fib

rin &

gr

anul

atio

n

Fibrin

(Blo

od

clut

ting)

Fibrin

&

Gra

nula

tion

Gra

nula

tion

(bui

ldin

g up

str

uctu

re fo

r ne

w

tissu

e)

Gra

nula

tion

&

Epith

elisa

tion

Epith

elisa

tion

(wou

nd c

losu

re)

Scar

ring

Silver Vacuum

Biosurgery Hyaluronic acid Cotton Alginate Hydrofibre Activecoal Foamed material (FM) Silicone Ev.Hydrocolloid Hydrocolloid

Wet

MIX 557 ??? Debridement Silver

Vacuum Biosurgery Hyaluronic acid

Cotton Alginate Hydrofibre Active coal FM Silicone Ev. Hydrocolloid Hydrocolloid

Moist

MIX 557 ??? Debridement Silver

Vacuum Cotton

Biosurgery Hyaluronic acid Film Impregnated gauze Hydrogel Silicone Hydrocolloid

Dry

Decubitus Ulcer

Diabetic foot

MIX 557 ???

Treatment of Infections (TIME)


Epidermide

Derma

BloodInfections Keloid Bad scar tissue

Applying MIX 557 dailyone obtains a perfect

time course for:

The inflammatory ph.

The proliverative ph.

The remodeling ph.

2° phase 3° phase1° phase

Presentation part B

Impact of the botanical insecticide NeemAzal® on

survival and reproduction of the biting louse Damalinia limbata on

Angora goats

This study was conducted to assess the insecticidal potential of NeemAzal®, a commercial product based on neem seed kernels forcontrolling Damalinia limbata on Angora goats and in particular todetermine its effects on the survival of adult and nymph life stages and to characterize its activity on the reproductivecapacity of female lice.

Objectives

Impact of NeemAzal on the survival of both the nymphal as the adult stages of Damalinia limbata

Evaluation of the impact of NeemAzal on the gonotrophic cycle by close study of the reproductiveorgans of female Damalinia limbata

Pediculosis of biting louse in high added value fibre animals

damage to the fibre due toscratching

Economic damage: both fibre quality as quantity are influenced

Pharmacological control (organophosphates, pyrethroids, avermectine, inhibitors of development)

Inconveniences: toxicity, resistance, residues

Biological cycle of Damalinia limbata

Egg

Adult

Nymph (3 stages)

♂ ♀

Experimental design

• 47 angora goats were available: – 27 females 0.5 to 5 years old, weighing 12.0 kg – 43.5 kg – 20 males 0.5 to 5 years old, weighing 11.5 kg – 56.5 kg.

• Goats were divided in 4 sex-matched treatment groups, each consisting of 6 heavily infested animals and 6 (5) moderately infested animals, determined on the basis of a pre-treatment survey. The animals were randomly allocated to the 4 treatment groups– Neem Azal® 650 ppm,– Neem Azal® 125 ppm, – Neguvon® (positive control) – Untreated control

Treatment of Angora goats

Treated with a nebuliser with a spray difusor

Dried in the shade

Treatments: groups of goats (N = 11) were sprayed either with 1 litre formulated Neem Azal® solution at 650 ppm or 125 ppm or with Neguvon® solution

Lice count on fleeceSix zones examined

Moment during count

Distribution of Damalinia limbata on the fleece Upper body region Lower body region Geom. mean (CI) % Geom. mean (CI) % Adult lice

12 (9-17)

27

32 (25-42)

73

Nymphs 40 (30-55) 61 26 (20-33) 39

Observed density in 60 cm2 = 50 – 150 lice(minimum value = 11 ; maximum value = 362)

Estimated density per animal (~1 m2) = 8.500 – 25.000

Control of D. limbata infestation on Angora goats

controlneguvon

NA 125NA 650

control neguvon

NA 125

NA 650

control neguvo

NA 125

NA 650

control

neguv

NA 125

NA 650

contro

negu

NA 12

NA 65

contr

neg NA 1

NA 6

cont

ne NA

NA

co

n N

N

weeks (before and after treatment)

0

50

100

150

200

250

300

350-1 0,5 2 5 10 14 18 22

nymphs adults NA treatmentsA Al 5 0 u 2 5 1 6 n

Fig. 1 Impact of Neem Azal® treatment on D. limbata adult and nymph densities during 22 weeks of follow up.

Distribution of D. limbata adults and nymphs on the upper and lower body regions of the goat host

before and after treatment

Fig. 2. A) pre-treatment distribution; B) louse population control phase: data pool of weeks 0.5 to 10 after treatment, in which lice densities were significantly reduced in both treatment groups; C) louse population re-establishment phase: data pool of weeks 14 to 22 after treatment.

p r e -t r

c o n t r o l

n e g u v o nN A 1 2 5

N A 6 5 0

c o n tr o l n e g u v o n

N A 1 2 5 N A 6 5 0

-4 0

-2 0

0

2 0

4 0

B CA

n y m p h sa d u l ts

N A tr e a tm e n ts

Tab. 2. Percent reduction of louse burdens observed at differenttime points after treatment during 5 months of follow up

Louse reduction (%)1) at different time points after treatment

Treatment 4 days 2 weeks 5 weeks 10 weeks 14 weeks 18 weeks 22 weeksNA650 36 84 s.2) 95 s. 96 s. 87 s. 76 s. -543)

NA125 44 71s. 86 s. 92 s. 60 s. 34 -153

Neguvon 91 s. 80 s. 73 s. 50 -35 -33 -159

1) Percent reduction calculated with reference to pre- and post-treatment

values of untreated and treated goat groups 2) s.= significantly different value (at P < 0.001) compared with that of the

untreated control group.3) Negative values are due to a higher number of lice observed at the

respective time point in respect to that at pre-treatment counts.

Percent control (Adult + Nymphs)

-40

-20

0

20

40

60

80

100

-7 4 13 30 60 90 120 150

Time after treatment

Perc

ent (

%)

Neem 625 ppmNeem 125 ppmNeguvon

Laboratory trialsTemperature = 36 ± 1°C

Relative humidity = ~70%

Fibre quantity = 0,022 g / tube

Dandruff = 1,6 – 8,0 mg / tube

Longivity tests on nymphs in the presence of dandruff treated in vitro

Tubes containing colonies ofD. limbata inside the incubator

Longivity tests on nymphs in the presence of dandruffcollected from treated goats (NeemAzal)

Evaluation of ovary morphology from female lice collected on treated Angora goat

0

5

10

15

20

25

30

35

11/07 15/07 17/07 21/07 23/07

Controllo NeemAzal 1250 ppm

Num

ber

of li

ve n

ymph

s

Measurement date

Impact of NeemAzal on nymph survival in in vitro

0

5

10

15

20

25

30

35

Controllo Solvente 125 ppm 625 ppm 1250 ppm

Presence of mature eggs Absence of mature eggs

N°

of in

divi

dual

sdi

ssec

ted

Impact of Neem Azal® on ovariole morphology and egg development in D. limbata females

displaying altered ovariole morphology

0

5

10

15

20

25

30

35

Controllo Solvente 125 ppm 625 ppm 1250 ppm

Absence of mophological alterationsPresence of morphological alterations

N°

of in

divi

dual

sdi

ssec

ted

Morphological alterations

yo

fc fl

oc

fc

nc

Vitellogenic follicle from neem treated female; The oocyte (oc) appears considerably reduced in size and detached from the follicular epithelium (fc) which appears flattened. The nurse cells (nc) have lost their tissue structure, acquired a rounded shape and invade the oocytecompartment. Vesicles of different size are accumulated in the oocytecompartment

Vitellogenic follicle of a control female; yolk spheres (yo) are present in the oocyte, that is surrounded by cuboid follicular cells (fc). Fold cells (fl), having differentiated from prefollicular cells, separate the nurse cell compartment from the ooplasm.

ConclusionsNeemAzal® reduces “fitness” (survival) of D. limbata both for the adult as the nymphal stage

NeemAzal® interferes with oogenesis

These observations correspond to many other studies carried out on insects of medical and/or veterinary importance (Diptera and Hemiptera

Louse infestation not only is controlled but the time periodprotection is active is protracted. This allows to use lesstreatments

Final considerations• Many more high added value applications exist

for Neem derived products• The main fields are medicine (both human as

veterinary), vector control and fertility control• All these application will necessitate neem

derived products of the highest quality and reliable composition

• These applications might justify the intense R&D and capital efforts that will have to becarried out in order to get to the market

Documents

High added value applications of Neem (Azadirachta indica ...neem.tea-nifty.com/neem/files/9.Dr.S. Andrew Van Der Esch.pdfPiodermitis Fistulae Rectum Prolapse Myiasis Bed sore or break