Lagenaria siceraria is a vegetable crop comprises two types of varieties; bitter variety and sweet variety

Synonymous name: Lagenaria vulgaris, Cucurbita lagenaria and Cucurbit a leucantha

English common name: Bottle gourd, white flowered gourd, ornamental gourd .

Vernacular name: Garaa alzeina (Sudan), Duppa (Saudi Arabia).

Botanical description : Annual, monoecious, long running or cl imbing, m usky - scented herb; stem robust, longitudinally furrowed, and pi lose with jointed, gland- tipped hairs; tendri ls usual ly bifid. Leaves with petio le 5-30 cm long with 2 pore-l ike glands at j unction with lamina, which is widely ovate, with cordate base with broad sinuses, dentate, lobes absent or 3-7 lobes or undu late, soft, with wh ite hairy beneath, 1 0-30 cm broad. Flowers axillary, sol itary, white in colour, opening at even i ng and close during the day.

In Sudan Lagenaria siceraria has not been known as a vegetable crop, the existing subspecies has been known as siceraria (Herwig Tepner, 2004) with a bitter fru it and hard shel l used as Domestic utensi ls l ike bottles, bowls, m i lk pots and containers of several types made out of the dried shel ls. (in Western Sudan), also it has been used for making stringed and wind musical instruments.

The targeted variety which had been used in the ointment is a sweet variety

Materials and Methods

Materials

Polyethylene glycol (PEG) ( Fluka AG chem ische Fabrik ) was obtained from pharmaceutics laboratory, col lage of pharmacy, Khartoum un iversity. Fucid i n (2 %) and Solcoseryl ointment 1 0% ( reference ointments used in wound heal ing ) were purchased from a pharmacy in Khartoum city

Plant material

The leaves were col lected from a field in Kassala city planted with lagenaria siceraria, the plant was identified and authenticated by taxonomist Abdul-Gapar Nasir Gomaa (Department of Biology, Faculty of Education, U niversity of Khartoum). the leaves were air dried in the shade then cleaned from impurities and coarsely ground using mortar.

Methods

Preparation of water extract

Water extract was prepared by repeated maceration with agitation using magnetic agitator. 30 grams of coarsely ground material of leaves of Lagenario siceraria were macerated in 200 ml distilled water for 4 hours. Extract was then fi ltered and the residue was macerated again using the same procedure, the fi ltrates were then col lected together and dried using freeze drier instrument.

Phytochemical screening of water extract

The general chem ical composition of the leaves of Lagenaria siceraria was determ ined by qual itative chem ical analysis by successive extraction with solvents of different polarities using the method of Teheknavorian and Wijesekera ( 1992 ). The leaves water extract declared presence of Polyuronide (muci l lage), glucides, reducing compounds, saponins and tannins

Antimicrobial activity of lagenaria siceraria water extract

For testing the antibacterial activity of Lagenaria siceraria leaves water extract against four standard bacteria wh ich were Staphylococcus aureus, Klebsiella pneumonia, Proteus vulgaris and Pseudomonas aeruginosa, the cup-plate agar diffusion method (Kavanagh, 1972) was used with some m inor mod ifications

For testing antifungal activity against Candida albicans, the same procedure as for bacteria was adopted. Instead of nutrient gar, Sabouraud dextrose agar was used. The inoculated medium was incubated at 25° C for two days

Lagenaria siceraria leaves water extract did not detected any activ ity against any of the tested organisms, the inhibition zone declared zero reading.

Ointment preparation

40 gm of 2% ( 1 % and 0.5 % also can be effective) of Lagenaria siceraria leaves water extract were prepared using polyethylene glycol (PEG) as a water soluble base (any su itable base can be use instead of PEG).

PEG was used as a m ixture of PEG crystal (m.wt.4000) and PEG solution (m .wt.400) 33.5%: 66.5% respectively, the mixture was stirred gently by infusion in a water bath (60-70°C) ti l l they are homogenously distributed, then the extract was added and stirred gently ti l l it completely d issolved in polyethylene glycol m ixture and the whole m ixture was cooled with continuous stirring. Acute oral and dermal toxicity of Lagenaria siceraria leaves aqueous extract

Acute toxicity was performed according to the World Health Organ ization (W HO) guideline (W HO 2000) and the Organization of Economic Co-operation and Development (OECD) guideline for testing of chemicals 420 (OECD 2001 ).

Acute oral toxicity

About 24 Swiss Wister albino rats weighing 1 80-250 gm were suppl ied by the National Experimental Animal House (N EAH), Medicinal and Aromatic Plants Research Institute (MAPRI), National Centre for Research (NCR ), M in istry of Science and Technology (MOST), Sudan. They were housed under standard environmental conditions under 12 hours dark-light cycle, and al lowed free access to drinking water and standard pel let diet. Rats were accl imated to hold ing faci l ities for 1 week prior to dosing. The rats were divided into 4 groups 6 rats in each group (3 males and 3 females). The animals were fasted over night with free access of water prier oral adm inistration. The three treated groups were orally given the aqueous extract in a single dose of 500, 1 000 and 2000 mg/kg body weight by gavage using gauged plastic syringe wh ile the control group received only water veh icle.

The an imals were monitored for apparent signs of toxicity and mortal ity after the adm inistration at the first, second, fourth and sixth hour and once dai ly for next 1 4 days .at the 1 5 ^th day after administration, al l rats were weighed and the blood samples for hematological and blood chem ical analyses were taken from common carotid artery after bei ng anesthetized by inhalation using d iethyl ether. A l l rats were sacrificed after the blood col lection, and then the vital organs incl uding heart, l ivers and kidneys were grossly and histopathological ly exam ined.

Acute dermal toxicity

Acute dermal tox icity was performed according to the World Health Organization (WHO) guidel ine (W HO 2000) and the Organization of Econom ic Co-operation and Development (OECD) guidel ine for testing of chem icals 404 (OECD 2002).

In order to reduce number of animals, on ly 6 Swiss Wister al bino rats were used (3males and 3 females). Females were null iparous and not pregnant. An imals were al lowed free access to drinking water and standard pel let diet.

Approximately 1 0 % of dorsal skin area of each rat was shaved without any abrasion 24 hour before the test.

After shav ing the animals were individual ly housed under standard env ironmental cond itions.

Approximately 0.5 gm of the tested substances (ointment of Lagenaria siceraria leaves water extract) at a dose of 2000 mg/kg body weight (lower limit of acute toxicity) was held in contact with the exposed skin under a gauze pad up to 4 hours. Following this the skin was washed with warm water. Untreated skin area was held as control.

The animals were then observed for any signs of toxicity during the observation period including irritation corrosion or any reaction at the application site, change in the fur, eyes and any other overt signs of toxicity. Body weight determination of each animal was done just prier tested substance application and then at dayl4. At day 15 all histopathological examination were done for the treated area of the skin.

Manual blood count

This include Haemoglobin (Hb), white blood cells (WBC), red blood cells (RBC) Neutrophils (N), Lymphocytes (L), Basophils (B), Monocyte (M), Eosinophils (E).

The blood collected from a common carotid artery in a test tube containing an anticoagulant (EDTA) to stop it from clotting, and transported to a laboratory.

Manual counting was used in this procedure in which Counting chambers that hold a specified volume of diluted blood (as there are far too many cells if it is not diluted) were used to calculate the number of red and white cells per liter of blood

To identify the numbers of different white cells, a blood film was made, and a large number of white cells (at least 100) were counted which gave the percentage of cells that were found in each type. By multiplying the percentage with the total number of white blood cells and so the absolute number of each type of white cell can be obtained.

Blood chemistry study

in which the serum was separated using centrifuged (Hittich EBA35) and was hold to the laboratory in eppendorf tubes and the levels of blood urea nitrogen (BUN), Creatinine (C EA), total protein (TP), albumin (ALB),total bilirubin (TBI), direct bilirubin, alkaline phosphatase (ALP), serum glutamic-oxaloacetic transaminase (AST), serum glutamic-pyruvic transaminase (ALT) and Na, K electrolyte were measured using automatic blood chemistry analyzer.

Histopathological examination of vital organs in different groups

After the blood collection, the animals were sacrificed for vital organs examinations which were liver, heart and kidney.

Preparation of the tissue for histopathological examination was carried out according to the method described by Durury and Wellington (1980). following grossly examination of the organs, they were washed with normal saline and then fixed in 10% buffered formalin solution; following fixation, the tissues were dehydrated with 90% ethanol then embedded in paraffin, cut into thin sliced section (using 0.5 microtome HM325 Germany), stained with haemotoxyline- eosin dye and observed under light microscope. Statistical analysis

Statistical package for social sciences programme (SPSS) version 1 0 was used for al l biological trials

Results of the acute toxicity

In acute oral toxicity, the animals were monitored for any signs of toxicity including mortal ity, reduction of body weight, changing in haematological profi le and blood chem istry and histopathology of the vital organs (l iver, heart and kidney).

The four group of animals which were G l : adm inistered on ly water, G ₂ : administered 500mg of L.s. leaves aqueous extract/ kg body weight, G3 : adm inistered l OOOmg L. s. leaves aqueous extract/ kg body weight, G4 : adm inistered 2000mg of L.s. leaves aqueous extract/ kg body weight and G5 : treated topical ly with polyethylene glycol containing L.s. leaves aqueous extract 2%.

Univariate analysis of variance of the acute toxicity of Lagenaria siceraria leaves aqueous extract; Blood analysis

i-Tests of Ber een-Subjects Effects

Dependent Variable Results

a. R Squared = .920 (Adjusted R Squared = .904) The Mean of different blood arameters in different treatments

Parameter

Univariate analysis of variance of the acute toxicity of Lagenaria siceraria leaves aqueous extract; Liver function

i-De endent Variable Result

i-Dependent Variable Result

. R Squared = .743 (Adjusted R Squared

The Mean of different liver function parameters in different treatments

Parameter Univariate analysis of variance of the acute oral toxicity of Lagenaria siceraria leaves aqueous extract; Kidney function

i-Tests of Between-Subjects Effects

dependant variable results

. R Squared = .996 (Adjusted R Squared

The Mean of different Kidney function arameters in different treatments

Parameter The four treated group didn't show any signs of toxicity during all of the observation period, no mortality or any reduction of weight has been registered during the experimental period. In topical treatment, no irritation, no corrosion or any signs of allergy or skin sensitivity against the ointment

Histopa hology of the vital organs of acute toxicity

(left): Section in liver of G3 (rats orally administered 1000 mg of L s. leaves aqueous extract) showing no changes in hepatocytes ( arrows) or blood sinusoids ( white arrow), CV ( central vein), (right): Section in rat heart of G3 showing dilated coronary artery . Increase space around vessel star). Slight changes in cardiac muscle (H&E x400).

Fig. (22) (left ): Section in rat kidney of G ₄ (rats received 2000mg of L s. leaves aqueous extract) showing marked Wood vessel congestion ( black star) atrophy of renal corpuscle( white star) . Slight degeneration of tubular cells( arrows), (right): ) section in rat liver G ₄ near portal area showing slight disturbance of hepatocyte cords( arrows) congestion of portal vein ( PV) and proliferation of bile duct ( white arrows) (H & E x 400)

Section in rat heart of G4 showing slight deformity of blood vessels (BV) . with slight loss of transverse striation( arrows)

Result of Dermal toxicity

In dermal toxicity, the rats treated with polyethylene glycol ointment containing L. s. leaves aqueous extract in a dose of 2000 mg kg body weight

Fig. (24): Sections from both control (C) and treated (T) rat skin notice that there are no signs of inflammation or irritation . The only change is slight increase in epidermal thickness , horny layer and dermal glands. Trials carried out to insure the effectiveness of the ointment

About three in vivo trials had been carried out to insure the effectiveness of Lagenaria siceraria leaves water extract ointment tow trials was in Swiss W ister albino rats of both sexes which were supplied by the National Experimental Animal House (NEAH), Medicinal and Aromatic Plants Research Institute (MAPRI), National Centre for Research (NCR ), Ministry of Science and Technology (MOST), Sudan, the third trial was clinical trial.

First in vivo trial: Effect of Lagenaria siceraria leaves aq ueous extract on infected wound healing

According to the model of Cross et al (1995) which is modified by Hemmati and Mohammadian (2000), and fulfill by AbdAlla (2004), an artificial wound measuring 2 cm . in diameter was made by excising the skin with in the border of the circle submitted by the metal object measuring 2cm. to the level of loose subcutaneous tissue.

Artificial infection was made using cl in ical isolate of Staphylococcus - aureus suspension using transferpette adjustable volume automatic microl iter pipette to spread 0. 1 m l . of the suspension in every wound so as to i nduce infected wounds.

After excision and infection, the rats were housed individual ly to prevent undesirable attack between wounded animals and they were divided into five groups each contain ing 6 animals:

Group 1 : infected wound only

Infected wound in this group were left without treatment (-ve contro l)

Group 2: infected wound + polyethylene glycol (+ve control)

I nfected wound of the animals in this group were treated with polyethylene glycol (water washable base) as a positive control starting from the first day

Group 3: infected wound + Lagenaria siceraria leaves aq ueous extract

In this group the infected wound of the animal was treated topical ly with polyethylene glycol containing 2% of Lagenaria siceraria leaves aqueous extract every 1 2 hours starting from the first day.

Group 4: infected wound + Fucidin ointment 2 %

The infected wounds in this group were treated topical ly with Fucidin ointment (2 %) as standard heal ing agent starting from the first day.

Group 5: infected wound + Solcoseryl ointment 10% The infected wounds in this group were treated with Solcoseryl ointment 1 0 % as standard heal ing agent starting from the first day.

Second in vivo trial: effect of L. sicerdria leaves aq ueous extract on diabetic infected wound healing

Induction of hyperglycemia (Diabetes)

Standard procedure (Kodama et al. 1 993) was adopted with some m inor modi fication to induce hyperglycemia in Swiss W istar Albino rats.

About 24 rats of both sexes weighing 1 1 0gms- 1 45gms were used in this trial. The rats were administered Alloxan dissolved in citrate buffer of pH 3.5 ( 1 75 mg /kg of the body weight). They were injected intraperitoneal ly.

Four rats died on the day after injection of Al loxan, five were d ied at the th ird day of injection.

G l ucose levels were measured three days after injection of Al loxan for the rest of the al ive rats ( 1 5 rats) in Central Laboratory of Khartoum Hospital .

At the beginning of the experiment only 10 rats remained.

Artificial infected wounds were made as described before in the infected wound healing experiment.

The rats were d i v ided into two groups each contained 5 rats as fol lows

Group l : U ntreated rats (control)

In this group the diabetic infected wounds were left without treatment

Group 2: Diabetic infected wound + aqueous extract of L. siceraria leaves 2% in polyethylene glycol

In this group the infected diabetic wounds were treated topical ly with an ointment of L. siceraria leaves aqueous extract 2% in polyethylene glycol every 1 2 hours starting from the first day

Third in vivo trail; Clinical trial

About 40 vol unteer (most of the wounds was diabetic wounds) had been cured by Lagenaria siceraria leaves water extract ointment.

Results of the three trials

First trial

In the first group (-ve control), the heal ing took about 1 2 days to be complemented whereas in the group treated with po lyethylene glycol ointment 2% (+ve control) the heal ing was completed within 1 1 days (the group treated with polyethylene glycol contain ing L. s. aqueous extract 2%), the period was dim in ished to 7 days whereas in group treated with Fucidin 2 % and Solcoseryl ointment 10% the healing was completed within 10 and 9 days respectively

Second in vivo trial

In untreated group (Groupl) the healing was completed within 14 days with the presence of pus cells during healing period whereas in the treated group, the healing period declined to 9 days to demonstrate intelligible effect of L. s. leaves aqueous extract on diabetic wound healing.

Third in vivo trial (clinical trial)

All the treated volunteers had been recovered, 100 % results was obtained. Some of the patients recovered within three days, others within 7 days whereas others take 10- 15 days.

Histological evaluation of wound healing treated by Lagenaria siceraria leaves water extract

Histological study of treated wound declared complete healthy epithelization with separation of wound scap (fig. 17).

The cells of the epidermis showed hyperplasia (increase in cell layers) near the edges of the wound which creeps in a normal thickness over granulation tissue which showed normal parallel orientation of collagen fibers and capillaries with few fibroblasts and inflammatory cells.

Section of untreated wounded rat skin (day 14) showing lacking of complete epithelization (black arrows). Presence of granulation tissue near the surface ( star), (b) Magnified power from the previous section showed the presence of immature granulation tissue (dotted square) of unhealed wound ( rich in blood capillaries (arrows) ), (c) section in control rat skin (nearby wounded area) showing the different layers ; epidermis (black arrow) consists of thin stratified squamous epithelium and dermis rich in collagen bundles( white stars) . Underlying skeletal muscles( (black star) could be seen separated from dermis by scanty adipose tissue (dotted arrows), (d) section from skin of control untreated wounded rat skin stained with Masson trichrome to show both original mature collagen( black star) and new immature fine fibrils separated by the high density of fibroblasts (pink nuclei- white arrows) (MassonX400 ( H&E x400).

(a) Low power of treated wounded skin by L s. leaves aqueous extract (9 days) showing complete epithelization( black arrows) . Notice the separated scab( star) .and the presence of few congested blood vessels (white arrow) ( H&E X4), (b) Section of rat wounded skin(9 days) after painting with L. s. leaves aqueous extract (2%) showing hyperplasia of epithelial epidermal layer( white star). Scar tissue lack skin appendages in deep dermis ( black star). Notice the numerous blood capillaries (black arrow ) in still immature granulation tissue. Normal collagen could be seen ( white triangle) ) ( H&E x 200), (c): section in rat skin nearby wounded area showing normal thickness of epidermis( arrow) with normal collagen( star) tissue around hair follicles and associated sebaceous glands( white arrows), (d): section in scar tissue of treated rat wound notice maturation of scar tissue with decrease in blood vessel density ( arrows) increase in mononuclear cells ( lymphocytes- white stars) compared to untreated.

Medicinal uses of the ointment

• Normal wound healing as well as diabetic wound healing

• Bums

• Allergy

• Irritation

• Give rigidity to those who has decaying in their outer layer of the skin References

Abdu Allah, AZ. (2004). Antimicrobial and wound healing activity of ten selected medicinal plants. A thesis submitted for the Degree of M.Sc. of the University of Khartoum (Microbiology).

Cross SE, Naylor IL, Coleman RA and Teo TC. (1995). An experimental model to investigate the dynamics of wound contraction. Br. J plastic surgery,48: 1 89-1 97.

Durury, R.A.B. and Wellington E.A.(1980).Carlton's Histopathological technique, 5 ^th ed. New York. Oxford university press.

Hemmati AA And Mohammadian F. (2000). An investigation into the effect of mucilage of quince seeds on wound healing in rabbit. J. herbs, spices and medicinal plants, 7: 41 -46.

Herwig Teppner (2004). Notes on Lagenaria and Cucurbita (Cucurbitaceae) - Review and new contributions. Phyton (horn, Austria), 44: 245-308.

Kavanagh F. (1972). Analytical microbiology. Academic press, New York and London, 1 1 : 1 1 .

Telieknavorian A. and Wijesekera R.O. (1992). Methodology the industerial for analysis of vegetable drugs, practical manual on

Organization for Economic Cooperation And Development. OECD. Guideline for Testing of Chemicals. Guideline 423. Acute Oral Tox icity- Acute Class Method. Adopted 17 Dec. 2001 . Available at:<http://iccvam. niehs.nih.gov/SuppDocs/FedDocs

OECD/OECD_GL423.pdf>.Accessed on: 10 dec. 2007.

Organization for Economic Cooperation And Development. OECD. Guideline for Testing of Chemicals. Guideline 404. Acute Dermal Irritation/Corrosion -Acute Class Method. Adopted 24 April 2002. Available at: < http:// www.oecd-ilibrary.org/test-no-404-51mqcr2k7pnp.pdf >

Name of inventor: ELAMIN Ihsan Mohammed Elhadi

Address: Tuti Island, Khartoum state, Sudan, Box no.1506, Po code: 1 1 1 1 1 , Elhadi Madam Mohammed Sudan News Agency, Gamhoria street, Khartoum, Sudan