July – September 2010 RJPBCS Volume 1 Issue 3 Page No. 750 Research Journal of Pharmaceutical, Biological and Chemical Sciences Wound healing… [603115]

ISSN: 0975 -858 5

July – September 2010 RJPBCS Volume 1 Issue 3 Page No. 750

Research Journal of Pharmaceutical, Biological and Chemical
Sciences

Wound healing activity of human urine in rats

Ramesh HA1, Mohammad A zmathulla2*, Malay B aidya2, Mohammed A sad1

1Department of Pharmacology, Krupanidhi College of Pharmacy, #5, Sarjapu r Road, Koramangala, Bangalore -560
034, INDIA.

2 Department of Pharmacology , Gautham College of Pharmacy, Sultan Palya, R.T. Nagar Post, Bangalore -560 032,
INDIA.

ABSTRACT

Urine is used traditionally in India for the treatment of burns and wounds. It is believed that applying
urine over the wound increases healing. The present study was carried out to evaluate the effect of human urine,
urea (2.5%), and urea (5%) on experimentally induced wounds in rats and compare the effects observed with an
antisep tic agent, povidine iodine solution. The models selected were excision wound, incision wound, burn wound
and dead space wound. In the excision wound and burn wound models, a significant decrease in period of
epithelization and wound contraction -50% was obs erved in all the treatment groups when compared to control
except low dose of urea (2.5%), which showed reduction in only period of epithelization. In the incision wound
model, a significant increase in the breaking strength was observed. Human urine treat ment orally produced a
significant increase in the breaking strength, dry tissue weight and hydroxyproline content in dead space wound
model. It was concluded that human urine applied topically or administered orally (10 ml/kg, p.o) possesses wound
healing activity.
Keywords : Human urine, incision wound, excision wound, dead space wound, burn wound.

*Corresponding author
E-mail: [anonimizat]

ISSN: 0975 -858 5

July – September 2010 RJPBCS Volume 1 Issue 3 Page No. 751

INTRODUCTION

The u se of urine for the trea tment of diseases is an ancient practice described in many
Hindu and Chinese literature. It is commonly called as auto -urotherapy, urotherapy or urine –
therapy. It is believed that urotherapy was also used by indigenous Americans and ancient
Egyptians [1]. Urine therapy may have been referenced in the Bible: “Drink waters from thy
own cistern, fl owing water from thy own well ” [Proverbs 5:15 ]. There are very few scientific
reports on the effect of urine. However, the uses of urine have been mentioned in ancie nt
literatures in different countries including India. In India, it is known as Amaroli, Shivambu or
Autourine therapy. This practice is derived from yoga wherein urine is treated as food,
medicine and an immune booster. Urotherapy is believed to be benefi cial for the treatment of
wide variety of disorders from sores to cancer . Urine is said to be effective against the flu, the
common cold, fever, broken bones, toothaches, dry skin, psoriasis and all other skin problems.
It is said to deter aging and may be helpful against AIDS, cancer, allergies, animal bites, asthma,
heart disease, hypertension, burns, fatigue, infertility, baldness, insomnia, gangrene, chicken
pox, tuberculosis, and a countless number of other diseases and disorders [2]. Urotherapy is
reported to be beneficial for the treatment of cancer [3]. The American Cancer Society
recommends urine therapy for the treatment of cancer [4]. It is also known to treat problems
related to skin and hair like acne, hair loss, warts, wrinkles and infections [5].

One of the important uses of human urine is for treatment of burns and wounds. It is
believed traditionally in India that applying urine over the wound increases healing. Saharan
Bedouins also use d urine to cleanse burns and wounds. Urotherapy for tr eatment of wounds is
also mentioned in the Ebers Papyrus of 1500 B.C, one of the oldest surviving documents of
Egyptian history [1]. The wound healing activity of urine is believed to be due to the presence of
urea, which is an effective antibacterial and antifungal agent [6].

Although, human urine has been recommended for the treatment of wounds from time
immemorial, there are no scientific reports to confirm the effect of urine on the wounds. The
present study was undertaken to evaluate the effect of eff ect of human urine on
experimentally induced wounds in rats.

MATERIALS AND METHODS

Experimental animals

Albino Wist ar rats of either sex weighing 200 -225 gm and albino rabbits weighing
between 1.5 to 2.0 kg were used. Animals were maintained under hyg ienic condit ions and they
were provided with commercial food p ellets and tap water ad libitum . Cleaning and sanitation
work were done on alternate days. Paddy husk was provided as bedding material, which was
changed everyday. The cages were maintained clea n and all experiments were conducted
between 9 am to 5 pm.

ISSN: 0975 -858 5

July – September 2010 RJPBCS Volume 1 Issue 3 Page No. 752

Chemicals

Ketamine injection was procured from Prem Pharmaceuticals Pvt. Ltd. [Indore, India]
and xylazine was from Indian Immunological Ltd. [Guntur, India] , hydroxyproline a nd
paradimethylamino benzaldehyde were procured from SD Fine Chemicals Pvt. Ltd. [Mumbai,
India ], sodium hydroxide, hydrogen peroxide and copper sulphate were purchased from Nice
Chemicals Pvt. Ltd. [Mumbai, India] , hydrochloric acid was obtained from Ranb axy Fine
Chemicals Pvt. Ltd. [Mumbai, India] .

Selection of dose and treatment period

First void u rine in the morning without any dilution was used for topical application in
excision, incision and burn wound models. Urine was applied to cover the entir e wounded area.
In dead space wound model, u rine [10 ml/kg] was administered orally . The treatment period
was 10 days for incision and dead space wound models and in case of excision and burn wound
models, the treatment was continu ed till the day of scar f alling.

Urine analysis

Qualitative urine analysis was carried out to detect the presence of normal and
abnormal constituents [7].Quantitative urea analysis was carried out every alternate day during
treatment by diacetyl monoxime method [8].

Excision wound [9, 10]

The animals were anesthetized by using k etamine [ 100 mg/kg, im ] and xylazine [ 16
mg/kg, im] [11] . An impression was made on the dorsal thoracic region 1 cm away from
vertebral column and 5 cm away from ear on the anaesthetized rat. The parti cular skin area
was shaved one day prior to the experiment. The skin of impressed area was excised to the full
thickness to obtain a wound area of about 500 mm2. Haemostasis was achieved by blotting the
wound with cotton swab soaked in normal saline. The a nimals were then grouped and treated
as follows: Group I: saline [ 0.9% w/v] . Group II: povidine iodine [5% v/v] . Group III: low dose of
urea in saline [2.5% w/v] .Group IV: high dose of urea in saline [5%] Group V: human urine .
Wound area was measured by tr acing the wound on a millimeter scale graph paper on
predetermined days i.e., 2, 4 , 6, 8, 10, 12, 14, 16, 18, 20 , 22 days post -wounding for
determination of wound contraction -50%. Falling of scar leaving no raw wound behind was
taken as end point of comple te epithelization and the days required for this was taken as period
of epithelization .

Incision wound [12, 14]

Para vertebral straight incision of 6 cm length was made through the entire thickness of
the skin, on either side of the vertebral column with the help of a sharp scalpel. After complete

ISSN: 0975 -858 5

July – September 2010 RJPBCS Volume 1 Issue 3 Page No. 753

homeostasis , the wound was closed by means of interrupted sutures placed at equidistance
points about 1 cm apart. Animals were treated daily with drugs, as mentioned above under
excision wound model from 0th day to 9th post -wounding day. The wound breaking strength was
determined on 10th day by continuous, constant water flow technique.

Burn wound [15, 16]

Partial thickness burn wounds were inflicted on overnight -starved animals under
ketamine [ 100 mg/kg, im ] and xylazine [ 16 mg/kg, im ] anesthesia by pouring hot molten wax at
80 0C. The wax was poured on the shaven back of the animal through a cylinder of 300 mm2
circular opening. The wax was allowed to remain on the skin till it gets solidified. Immediately
after the injury and on subsequent days, the drugs or vehicle was appli ed topically as
mentioned above .

Dead space wound model [17]

This type of wound was created by implanting subcutaneously a 2.5×0.5 cm
polypropylene tube in the lumber region of dorsal side in anesthetized rats. Animals received
one of the following treatments from 0th day to 9th post wounding day.

Group I: control group: animal of this gro up received saline [ 1 ml/kg , p.o].
Group II: low dose of urea [250 mg/kg, p.o].
Group III : high dose of urea [500 mg/kg, p.o].
Group IV: human urine [ 10 ml/kg, p.o].

On the 10th post wounding day, granulation tissue harvested on the implanted tube was
carefully dissected out along with the tube. The tubular granulation tissue was cut along its
length to obtain a sheet of granulation tissue. The breaking strength was measured as
described under incision wound model. The pieces of granulation tis sue were collected, dried at
60 șC for 24 h to get a constant weight and weighed. The tissue was then used for the
determination of hydroxyproline content [18].

Skin irritation study [19]

This study was carried out on rabbits. The skin of the animal was shaved at four
different positions on the dorsal side, each about 6 cm in length [approx] . The first area was
kept as control , to which vehicle was applied. To the second area , human urine was applied .
The third area was treated with low dose of urea in saline [2.5%] and the fourth area was
treated with high dose of urea in saline [5%] . After 4 h , the skin wa s observed for signs of
inflammation and scored as follows: No erythema, no oedema -0; very slight erythema, very
slight oedema -1; well defined erythema, slight oedema -2; moderate to severe erythema,
moderate oedema -3; severe erythema, severe oedema -4. The study was carried on two
different animals and average of the two scores was taken as an index of skin irritation.

ISSN: 0975 -858 5

July – September 2010 RJPBCS Volume 1 Issue 3 Page No. 754

STATISTICAL ANALYSIS

Results ar e expressed as mean ± S EM. The difference between experimental groups w as
analyzed using one-way Analysi s of Variance [ANOVA] followed by Bonferroni test and were
considered statistically significant when P<0.0 5.

RESULTS
Urine analysis

Qualitative analysis of the human urine revealed the presence of following normal
constituents ; chloride , ammonia, urea, c reatinine , and uric acid . The abnormal constituents of
urine such as bile salts, ketone bodies and reducing sugar were absent. The urea content in the
urine samples during treatment was in the range of 3.9 g/dl to 6.9 g/dl.

Effect on excision and incision wound

A significant decrease in period of epithelization was observed in all the treatment
groups when compared to control. Comparative analysis revealed that human urine , urea [5%]
and povidine iodine had almost equal wound healing activity. There was a significant reduction
in wound contraction -50% in all the treatment groups except low dose of urea [2.5%] [ Fig.1]. In
incision wound model, the breaking strength of 10 day old wound was significantly increased by
all treatments when compared to control [Fig. 2].

Effect on burn wound

Like excision wound, application of human urine , urea [5%] and povidone iodine
topically shortened the period of epithelization and wound contraction – 50% significantly when
compared to control while the low dose of urea [2.5 %] was effective only in reducing the period
of epithelization [Fig. 3].

Effect on dead space wound

The breaking strength of 10 days old granulation tissue was significantly promoted by
human urine [10 ml/kg, p.o ], high dose of urea [ 500 mg/kg, p.o] and low dose of urea [250
mg/kg, p.o] . The dry tissue weight and hydroxyproline content of granulation tissue was also
increased significantly by all the treatments except low dose of urea [2.5%] [ Table I].

Skin irritation study

There was no evidence of any noticeable inflammation when h uman urine and urea was
applied over rabbit’s skin indicating that it does not possess any irritant effect.

ISSN: 0975 -858 5

July – September 2010 RJPBCS Volume 1 Issue 3 Page No. 755

Table I : Effect on breaking strength, dry tissue weight and hydroxyproline content in dead space wound model.

Treatment Breaking strength Dry tissue weight Concentration of
(ml) (mg) Hydroxyproline
(mg/g)

Saline (1ml /kg, po) 227.50 ± 3.594 76.60 ± 4.539 0.2566 ± 0.0133
Urea
(250 mg/kg, po) 278.33 ± 10.138* 92.16 ± 6.26 0.2683 ± 0.0164
Urea
(500 mg/kg, po) 431.66± 12.758*** 133.75 ± 3.83*** 0.3400 ± 0.0158*
Human urine
(10 ml/kg, po) 455.00 ± 12.111*** 136.50 ± 10.90*** 0.3466 ± 0.0152**

All values are mean + SEM, n=4 – 6, *P<0.05, **P<0.01 ***P<0.001 Vs control.

DISCUSSION

The result of the present study show that human urine possesses good wound healing
activity and it subs tantiates the traditional belief that human urine promotes healing of
wounds. The work was carried out using different models of experimental wounds to evaluate
the effect on breaking strength, epithelization and collagenation of wounds. Urea, which is the
main constituent of urine was used at two different doses because the reported normal value
of ure a is human urine is around 2.5 g/dl [8] while the urea content of the urine used in present
study was around 5 g/dl.

Collagenation, wound contraction and e pithelization are crucial phases of wound
healing. An intervention into any one of these phases by drugs leads to either promotion or
depression of the collagenation phase of healing. Growth hormone is known to promote the
healing process by enhancing epit helial cell proliferation and collagen formation. The collagen
synthesis is stimulated by various growth factors [20]. Growth hormone is also known to
promote the proliferation of fibrobla sts [ 21] and fibroblast proliferation form the granulation
tissue. I n the dead space wound model, oral treatment with human urine increased the
breaking strength of granulation tissue. The human urine contains many growth factors [ 22]
and the effect on collagen synthesis may be due to the presence of these growth factors.
Furthermore, urea is also reported to enhance co llagen deposition in wounds [23] . Hence, the
effect of urine on collagenation may be due to both presence of growth factors and urea in
urine.

Lipid peroxidation is an important process of several types of injuries like burn wound ,
inflicted wound and skin ulcers. Agents that inhibit lipid peroxidation are believed to increase
the viability of collagen fibrils . Several antioxidants such as vitamin C, metronidazole and
vitamin E are reported to increase the w ound healing [24]. Urine has good antioxidant effect
[25]. Hence, it can be suggested that the wound healing activity of urine may a lso be due to its
antioxidant activity.

ISSN: 0975 -858 5

July – September 2010 RJPBCS Volume 1 Issue 3 Page No. 756

Figure 1 : Effect on period of epithelization and wound contraction in excision wound model
***** ********* ***
02468101214161820
Control Povidine
iodine (5 % v/v) Urea
(2.5 % w /v) Urea
(5 % w /v)Human urine
TreatmentWound contraction-50 % & Period of
epithelization (Days)Wound contraction-50%
Period of epithelization

All values are mean + SEM, n=6, *P<0.05, **P<0.01, ***P<0.001 Vs control.

Figure 2 : Effect on breaking strength in incision wound model.
***
**** ***
050100150200250300350400450
Control Povidine
iodine (5 % v/v) Urea
(2.5 % w/v) Urea
(5 % w/v)Human urine
T rea tmentB reaking S treang th (ml)

All values are mean + SEM, n=6, *P<0.05, ***P<0.001 Vs control.

Figure 3 : Effect on period of epitheliza tion and wound contraction in burn wound model.
*** *** ****** ****
02468101214161820
Control Povidine iodine
(5 % v/v) Urea
(2.5 % w /v) Urea
(5 % w /v)Human urine
Treatment Wound contraction-50% & Period of
epithelization (Days)Wound contraction-50%
Period of epithelization

All values are mean + SEM, n=6, *P<0.05, ***P<0.001 Vs control.

ISSN: 0975 -858 5

July – September 2010 RJPBCS Volume 1 Issue 3 Page No. 757

Earlier studies carried out on human urine suggest that human urine has good
antibacterial activity [26]. The antibacterial effect is report ed to be due to osmolality, urea
concentra tion and ammonium concentration. Out of these, u rea concentration was more
important determinant of antibacterial activity than osmolality or ammonium concentration. In
the present study, the effect of urea [5%] and human urine were almost similar. So, it is
speculated that the wound healing activity of human urine may be due to combination of its
antibacterial, antioxidant and growth promoting effects.

The human urine did not produce any skin irritation study ind icating that is safe for local
application. To conclude, human urine possess good wound healing activity when applied
locally or administered orally. The effect observed is similar to that produced by urea.

CONCLUSION

The results are the present study s how the human urine affects wound healing in
experimental animals. It showed a significant decrease in period of epitheliazation and wound
contraction in excision and burn wound model, an increase in breaking strength of 10 day
wound in incision wound mode l and 10 day old granulation tissue, dry tissue weight and
hydroxyproline content were significantly increase in dead space wound model. From the
above results it was concluded that human urine has significant wound healing activity in
excision, incision, burn and dead space wound model.

REFERENCES

[1] http://www.heartlandhealing.com/pages/archive/urine_therapy/index.html
Retrieved on 26.09.2006, 11.49 AM IST.
[2] http://curezone.com/forums/fm.asp?i=124372 . Retrieved on 26.09.2006,
11.55 AM IST.
[3] Eldor J . Urotherapy for patients with cancer. Med Hypotheses . 1997 ; 48(4):309 –
15.
[4] http://www.can cer.org/docroot/ETO/content/ETO 5_3X_Urotherapy.asp?siteare
a=ETO . Retrieved on 23.09.2006, 10.55 AM IST .
[5] http://www.dailynewarticles.com/article/201/20327/Urotherapy_A_Treatment_
With_The_Help_Of_Urine.html . Retrieved on 22.09.2006, 11.55 AM IST .
[6] Gloor M, Reichling J, Wasik B, Holzgang HE. Res Compleme nt Class Nat Med
2002; 9: 153 -159.
[7] Rajagopal G, Thoora BD. Practical biochemistry. 1st ed. New Delhi: Ahuja book
company Pvt Ltd; 2001; 39-42.
[8] Rajagopal G, Thoora BD. Practical biochemistry. 1st ed. New Delhi: Ahuja book
company Pvt Ltd; 2001;89 -90.
[9] Kamath JV, Rana AC, Chowdhury AR. Phytother Res 2003 ; 17: 970 -972.
[10] Morton JJ, Malone MH. Arch Int Pharmacodyn Ther 1972; 196: 117 -126.

ISSN: 0975 -858 5

July – September 2010 RJPBCS Volume 1 Issue 3 Page No. 758

[11] Vogel HG. Drug discovery and evaluation, 2nd ed., Springer -Verlag Berlin
Heidelberg, Berlin, 2002.pp: 1739.
[12] Lee KH. J Pharmacol Sci 1968 ; 57:1238 -1240.
[13] Ehrich HP, Hunk TK. Ann Surg 1969 ; 170 : 203 -206.
[14] Somayaji SN, Jacob AP, Bairy KL. Indian J Exp Biol 1995 ; 33(3):201 -204.
[15] Holla RK, Sequeria RP, Kulkarni DR. Indian J Exp Biol 1998 ; 26: 869 -873.
[16] Rao CM, George KM, Bairy KL, Somayaji SN. Indian J Pharmacol 2000 ; 32: 282 –
287.
[17] Padmaja PN, Bairy KL, Kulkarni DR. Fitoterapia 1994 ; LXV : 298 -303.
[18] Neuman RE , Logan MA. J Biochem 1950; 186: 549 -552.
[19] OECD GUIDELINE FOR THE TESTING OF CHEMICALS. Acute Dermal
Irritation/Corros ion. Available from URL: http://ecb.jrc.it/documents/Testing –
Methods/ANNEXV /B04web2004.pdf.
[20] Corton SR, Kumar V , Collins T . Robbins Pathologic Basis of Disease . 6th Ed. New
Delhi: Harcourt Limited ; 2003 : 96-111.
[21] Williams T C, Frohman LA . Pharmacotherapy 1986; 6: 311-318.
[22] Starkey RH, Cohen S, Orth DN. Science 1975; 189: 800 – 802.
[23] Telgenhoff D , Lam K , Ramsay S , Vasquez V , Villareal K , Slusarewicz P , Attar P ,
Shroot B . Wound Repair Regen 2007 Sep -Oct; 15(5): 727-35.
[24] Rao C M, Ghosh AIndian J Pharmacol 1997; 29: 29.
[25] Kirschbaum B. Clin Chim Acta 2001; 305: 167-173.
[26] Donald Kaye J. Clin Invest 1968 ; 47(10): 2374 –2390.