INTRODUCTION:

The seeds of Nigella sativa Linn. (Fam. Ranunculaceae), commonly known as black cumin, have been traditionally used for treating ailments like asthma, bronchitis, rheumatism and related inflammatory diseases. It has also been used to increase the production of milk in nursing mothers. It promotes digestion and to fight parasitic infections. It has been used to treat skin conditions such as wounds and eczema.¹

Various crude and purified components of the seeds were reported to exhibit multifarious pharmacologic actions like antihistaminic², antihypertensive³, hypoglycemic⁴, anti-inflammatory⁵, anti-neoplastic⁶, antifungal⁷, antibacterial⁸, antioxidant⁹, etc. Antimicrobial agents arrest the growth or destroys the microbial organisms in the wound. Inflammation will not stop until the wound becomes sterile.

Inflammation plays roles in fighting infection and induces the proliferation phase and it is a necessary part of wound healing process. However, if inflammation lasts too long it may cause tissue damage.¹⁰ Thus the reduction of inflammation is frequently a goal in therapeutic settings after the wound become sterile by anti-microbial action. Thymoquinone is a potent inhibitor of leukotrienes a mediator of inflammation, thus produce anti-inflammatory action.¹¹ Lipid peroxidation is a process that is found to be involved in different types of injuries like burn, inflicted wound and skin ulcers. Any drug that inhibits lipid peroxidation can save the collagen fibrils, increasing the strength of collagen fibers, thus preventing the cell damage.¹² It is reported that antioxidants, such as metronidazole, vitamin C, vitamin E promoted wound contraction and epithelization.¹³ Nigella sativa oil, dihydrothymoquinone and thymoquinone act as superoxide anion scavengers. They also act as general free radical scavengers.¹⁴ These components help in reducing lipid peroxidation.

Thus the above three broad actions, namely, anti-microbial, anti-inflammatory and antioxidant effects raise the expectation of obtaining appreciable wound healing effect with the oil, extracts or active constituents of Nigella sativa seeds. In majority of the reports of pharmacologic and therapeutic activities of Nigella sativa, the seed oil, its extracts or active constituents were administered orally as oil¹⁵ or by intra-peritoneal injections¹⁶. Norwood prepared a sustained release delivery system of thymoquinone to study its effect in colon cancer.¹⁷ It is evident that very little work has been done on topical delivery systems and oral formulations of Nigella sativa extracts or its active ingredients.

MATERIALS AND METHODS:

Animals

Albino Wister male rats weighing between 150 and 200 gm were used for the study in different models. Rats were housed in groups of six in polyethylene cages (32 x 24 x 16 cm) and were provided with commercial food pellets and water ad libitum. On every alternate days cleaning and sanitation works were done. Paddy husk was used as bedding material, which was changed every day. All the experiments were conducted between 10 am to 6 pm.

Approval of Institutional animal Ethics Committee (IAEC, Regn. No.1502/PO/A/11/CPCSEA) of Netaji Subhas Chandra Bose Institute of Pharmacy was taken for the animal experiment protocols.

Chemicals:

Zylazine (zylaxine 23.22 mg/ ml) was purchased from Indian Immunologicals Ltd, Andhra Pradesh; Ketamine (Hypnoket 500mg/10mL) was purchased from Chandra Bhagat Pharma Pvt. Ltd, Mumbai; Hydroxyproline, paradimethylamino benzaldehyde were purchased from S. D. Fine Chemicals Pvt Ltd., Mumbai; sodium hydroxide (NaOH) AR, hydrogen peroxide (H₂O₂), copper sulphate (CuSO₄) were procured from Merck Specialties Pvt. Ltd., Mumbai. n-Propanol, sulphuric acid (H₂SO₄) were procured from International Chemicals, Kolkata; hydrochloric acid (HCl) was obtained from Bharti Chemical Works, Kolkata; light liquid paraffin, carboxymethylcellulose (CMC), polyethylene glycol 400 and 4000 (PEG) were obtained from Loba Chemie, Mumbai and sodium alginate was obtained from Thomas Baker, Mumbai. Placentrax gel formulation was purchased from Albert David Limited, Kolkata; Povidone Iodine from Leben laboratories Pvt. Ltd., Mumbai.

Extraction of volatile oil from Nigella sativa seeds:

The seeds of Nigella sativa were collected from the local market of Chakdaha, Nadia, W.B. Randomly selected seeds of Nigella sativa were identified by the Macroscopic and Microscopic evaluation methods prescribed in The Ayurvedic Pharmacopoeia of India, Part-I, Vol -1 p157.¹⁸ The seeds were air dried under shade for 10 days in the pharmacology lab of Netaji Subhas Chandra Bose Institute of Pharmacy. The dried seeds were powdered in kitchen mixer to get a coarse powder. The seed-powder was taken in a separating funnel (capacity 500ml). The separating funnel was fitted on a round bottom flask containing boiling water. The flask was placed on a heating mantle. The reflux condenser was fitted on the standard joint of the separating flask. Steam generated in the round bottom flask was passed through the seed-powder and the volatile oil carrying steam was condensed and collected in a collecting tube. The reflux was carried out for 2 hours. The crude volatile oil with the steam condensate was taken in a Clavenger’s apparatus for final separation of the volatile oil.

Preparation of Topical ointment:

PEG 4000 is a solid waxy material and PEG400 is a liquid. Simultaneous melting of PEG4000 and PEG400 in 50:50 ratio at 55⁰C on a water bath followed by cooling produced the ointment base. Volatile oil of N. sativa oil was weighed accurately and levigated with the cooled ointment base on an ointment slab to obtain a uniform ointment (NSOINT).

Acute oral toxicity study:

The procedure of acute oral toxicity study was followed as per the method given in Office of Prevention, Pesticides and Toxic Substance (OPPTS) guidelines following Up and Down Procedure^{19, 20}. Male Albino Wistar rats (200-250g) were maintained under controlled animal house conditions. Access to food and water were provided ad libitum. The rats were acclimatized for 5days and fasted overnight. Food was withheld but not water. Animals were weighed and subsequently ‘limit test’ and ‘main test’ were performed.

Limit test:

The limit test was carried out with a dose of 5000 mg/kg body weight for one animal and if the animal died, main test was performed. If the animal survived, two more animals were dosed. If both survived, the test was terminated.

Main test:

The main test was performed with single ascending doses in the order of Di = 175, 550, 2000 and 5000 mg/kg body weights as per the Scheme depicted in Figure 1. D_i is the dose at i-th iteration.

Initially one animal was administered with 175mg/kg body weight [D_i] orally. If animal dies at D_i dose then main test is terminated. If animal survives, then two more animals are dosed and observed after 48hrs. If the animals die, two more animals are dosed and observed for confirmation. If both the animals survive then the next higher dose was tried. In this iteration the last dose that produced death of maximum 2 animals out of 5 was taken as the toxic dose.

Excision wound model:

Wister male albino rats weighing between 150-200 gm bodyweight were divided into five groups, each group consisting of 6 rats and each animal was kept separately under laboratory condition. They had free access to commercial pellet diet and water ad libitum. The groups were assigned different treatments as per Table 1.

Table 1 Various treatment groups for excision wound healing model

Group	Treatment
Group-I: Control	Animals received PEG ointment base
Group-II: Standard-I	Animals received Placentrax gel formulation
Group-III: Standard-II	Animals received Povidone Iodine
Group-IV: Test Group-I	Animals received Nigella oil (5%) in PEG Ointment (NSOINT-5) formulation
Group-V: Test Group-II	Animals received Nigella oil (10%) in PEG Ointment (NSOINT-10) formulation

The animals were anesthetized by using ketamine 40mg/kg rat body weight (rbw) and Xylazine, 10mg/kg rbw intraperitoneally. A circular impression was made on the dorsal thoracic region of anaesthetized rat, position of which was 1 cm away from vertebral column and 5 cm far from ear. The particular skin area was shaved one day before the experiment. The skin of marked area was excised to the full thickness to obtain a wound area of about 500 sq mm.^{21, 22}Haemostasis was achieved by blotting the wound with cotton swab soaked in normal saline.

Contraction that contributes to wound closure was studied by tracing the raw wound. Wound area was measured by scanning the traced area along with a piece of square paper of standard size (100 mm²) with CanoScan Lide N676U at 200dpi resolution. A software (AREACALC Ver 1.0) was prepared with Microsoft Visual Basic 2010 Express to measure the area enclosed by any curved line. The standard area was pasted in the software and the area value (A_std) was determined. The test area was pasted in the software and the area value (A_test) was determined. Finally the area (in mm²) of the irregular shape was determined by the following formula:

Area of the wound (mm²) =

A_test

---------- X 100 mm² Eqn. 1

A_Std

Wound area was measured on 2, 4, 8, 10, 12, 14, 16, 18, 22 days or till scar had fallen. Epithelialization was assumed to be complete when scar falls, leaving not raw wound behind, and the days required for this was taken as the period of epithelialization.

Incision wound model:

Male Wister albino rats, weighing between 150-200 gm bodyweight were divided into five groups. The animals were anesthetized by using ketamine 40mg/kg rbw and Xylazine, 10mg/kg rbw intraperitoneally Para vertebral straight incision of 6 cm length each were made through the entire thickness of the skin, on either side of the vertebral column with the help of a sharp blade. After complete haemostasis the wound were closed by means of interrupted sutures placed at equal distance, about 1cm apart. Animals were treated daily with drugs, from 0^th day to 9^th after wound was created. Finally the wound breaking strength was estimated on 10^th day on anaesthetized animals by continuous, constant water flow technique.²³ Allis forceps were firmly applied on adjacent normal skin on either side of incision wound at 3 mm away from the wound margin. The forceps on one side was hooked to a fixed metal rod while the other forceps were attached to a thread suspended by weights running over a pulley. As soon as gapping of the wound occurred, further addition of weights was stopped and immediately the weights were lifted so as to avoid opening of the entire wound. The weight required to produce gapping was noted.

Dead space wound model:²⁴

Wister male albino rat weighing between 150-200 gm body weigh were divided into three groups, each group consisting of 6 rats, and each animal kept separately under laboratory condition. The rats were given commercial pellet diet and water ad libitum. The groups were assigned different treatments as per Table 2.

Table 2 Various treatment groups for dead-space wound healing model

Group	Treatment
Group-I: Control	Animals of this group received 2% Tween 80 solution
Group-II: Test Group-I	Animals of this group received Nigella oil (250mg/kg rbw) per orally.
Group-III: Test Group-II	Animals of this group received Nigella oil (500mg/kg rbw) per orally.

This type of wound was created by implanting subcutaneously a 2.5 x 0.5 cm polypropylene tube in the lumber region of dorsal side. Animals received treatment as per their groups from 0 day to 9^th post-wounding day, on the 10^th post-wounding day. Granulation tissue on the implanted tube was harvested carefully. The tubular granulation was cut along its length to obtain a sheet of granulation tissue. The breaking strength was measured as per the method described in incision wound model. The pieces of granulation-tissue were collected, dried at 60^oC for 24 hours, to get a constant weight and then weighed. The dry granulation tissues were used for determining of hydroxyproline content.

Hydroxyproline Determination:

The granulation mass, dried in a hot-air oven at 60⁰C for about 24 hours. The dried mass was weighed and placed in a sealed tube containing 10 ml of 6N hydrochloric acid (HCl). The dry tissue weight (W₀) was taken as a parameter for analysis. Heating the sealed tubes at 110°C for 24 hours hydrolyzed the tissues. The hydrolysate was cooled followed by neutralization of the excess acid with 10N sodium hydroxide (NaOH) employing methyl red as indicator. The volume of neutralized hydrolysate was made up to 20 ml with distilled water. From this 0.1 ml was used to estimate hydroxyproline as per the steps give below²⁵.

1. Hydrolysate sample, 0.1 ml, was pipetted out into clean test tubes. Volume was made upto 0.5 ml with distilled water.

2. From the stock solution of standard hydroxyproline, 1.6 ml was transferred to a volumetric flask and diluted up to 100 ml. From this 0.5ml (equivalent to 8 μg) was pipetted out into a clean test tube.

3. To this 1 ml of 2.5 N NaOH, 0.01 M copper sulphate (CuSO₄) and 6% hydrogen peroxide (H₂O₂) were added. The tubes were placed immediately in a water-bath maintained at 80⁰C for 16 minutes and then cooled for 5 minutes.

4. To this 2 ml of freshly prepared 5% solution of paradimethylamino- benzaldehyde in n-propanol, and 4 ml of 3N sulphuric acid (H₂SO₄) were added. Test tubes were once again placed in a hot water-bath maintained at 80⁰ C for 15 minutes followed by cooling for 5 minutes.

5. The absorbance of the pink colour of these test samples were compared with standard hydroxyproline of known concentration samples at 540 nm using the following formula.

Histology of healed wound tissue:

The harvested granulation tissues from the implanted tubes were collected on the 10^th post wounding day of the experiment for the histopathological study. Samples were fixed in 10% buffered formalin, processed, blocked with paraffin, then sectioned into 5μm sections, and stained with hematoxylin and eosin. The staining was carried out by application of hemalum, a complex formed from aluminum ions and hematein, which is an oxidation product of haematoxylin. Hemalum colors nuclei, granules and calcified material as blue to purple. Eosin is an acid aniline dye. It will bind to and stain basic structures such as cationic amino groups on proteins. It stains them pink.

Skin irritation study:

The NSOINT was designed to be applied on wounded skin. It may produce irritation on immediate, prolonged or repeated contact with intact or abraded skin that may induce local inflammatory reaction if not corrosion. Therefore, primary irritation index of control, NSOINT-5 and NSOINT-10 were determined as per the test methods described in 16 CFR 1500.41²⁶. The rabbit was first immobilized in a rabbit holder. Hair of six areas (approximate area 1.5in x 1.5in each) on the dorsal side were clipped free of hair. Out of six areas three areas were abraded by rubbing with a piece of gauze gently. Control or formulation, approximately 500mg, was weighed and placed on a one sq.in. aluminium foil. The foil was then pressed on the test area as a patch. The patches were secured with the help of adhesive tape. On the first pair of areas only vehicle of the ointment was applied to serve as control. Second pair of areas were applied with NSOINT-5 and the third pair of areas with NSOINT-10. All the areas were covered with impermeable aluminium foils to reduce the drying of the vehicle. After 24 hours and 72 hours the test areas were uncovered and observed for erythema and edema. Skin irritation values of each pairs of test areas were scored and finally the “Primary Irritation Indices” of Control, NSOINT-5 and NSOINT-10 were calculated as per the calculation method described in 16 CFR 1500.41.

RESULTS AND DISCUSSION:

Acute oral toxicity study in rats:

Table 3: Acute oral toxicity data of volatile oil of N. sativa in rats

Dose (mg / kg rbw)	5000	175	550	2000
Animal Died	√	–	–	–
Animal Survived	–	√	√	√

Limit test was carried out on one male albino Wistar rat at 5000mg/kg rbw. The animal died which was followed by main test. In the latter test the animals survived in highest 2000mg/kg rbw dose. Therefore, it may be concluded that 2000mg/kg rbw was the safe dose.

Study of excision wound model with PEG ointment:

In this model epidermis and dermis are completely removed to the depth of subcutaneous fat or fascial planes. Healing occurs both from the margins and the base of the wound. It involves the formation of a fibrin clot where granulation tissue invades. This study is essential to evaluate the period of epithelialization.

From the permeation study, PEG-ointment was found to show best release property. Hence, PEG ointment of volatile oil [NSPEG] was taken as the test formulation in excision wound healing study. The study was conducted taking PEG ointment base as control, Placentrex gel and Povidone Iodine as standard drugs. Placentrex gel promotes growth in wounds while Povidone Iodine is a non-specific antimicrobial agent. Thus the activity of the NSPEG was compared with two standard agents having different mechanisms of wound healing activity.

Low and high dose of volatile oil containing PEG ointments were applied on the excised wound. All the studies were replicated with 6 animals each. Two parameters were determined (i) wound area and (ii) days of scar falling. From the wound area versus time (in days) two more parameters were calculated (i) time required for 50% wound contraction (WC₅₀) and (ii) mean wound healing time (MWHT).

The WC₅₀ was calculated using MS Excel as per the following formula.

Time	Area
0 day	A₀= known
t₁ = known	A₁= known
WC₅₀ = unknown	0.5A₀ (50% A₀)
t₂= known	A₂= known

From the time versus wound area data the 50% of A₀ was calculated. Then t₁, t₂ and corresponding A₁ and A₂ were selected which were known. The following formula was used to calculate the WC₅₀ from the above data.

Calculation of WC₅₀ assumes that the relationship between time and wound area is linear, which is more often not true. Although most of the workers are satisfied in reporting the wound healing parameter in the form of WC₅₀ it required to look at the relationship beyond the WC₅₀.

A non-parametric method was applied to determine another parameter that will adequately describe the wound healing data is the mean wound healing time that was determined from statistical moment theory. Just like Mean Dissolution Time (MDT) in case of drug release data, Mean Residence Time (MRT) in case of pharmacokinetic data, etc. a new parameter Mean Wound Healing Time (MWHT) was determined from the wound healing data. In this case the total time scale, till the scar falling day, was taken into consideration.

The procedure adopted was as follows:

(i) First the wound area (Y) versus time (X) were plotted. The area under the curve of Y vs. X was calculated by trapezoidal rule in MS Excel to get the AUC.

(ii) Second the wound area x time (i.e. XY) was plotted against time (X). This is called the first moment curve. The area under the first moment curve (AUMC) was calculated similarly by trapezoidal rule in MS Excel.

(iii) Finally, the MWHT was calculated from the formula

MWHT is a time term expressed in days and may be defined as the mean time required to heal the wound.

The confidence limit calculations were done with GraphPad Prism software. One way ANOVA in Graph Pad Prism software provided the significance of the hypotheses.

Table 4 Wound healing parameters obtained from excision wound model

Parameter studied

Epithelialization

period (days)

50% Wound contraction (days)

MWHT (days)

Control

(PEG ointment)

21.00

± 1.41

7.765

± 0.16

4.58

± 0.04

Placentrax gel

12.33

± 0.21*

4.128

±.29*

2.818

± 0.10*

Povidone Iodine

15.166

± 0.30*

6.306

± 0.10*

3.596

± 0.054*

N S (5%) in PEG ointment

13.83

± 0.16*

4.831

± 0.14*

3.235

± 0.03*

N S (10%) in PEG ointment

11.5

± 0.34*

3.868

± 0.16*

2.523

± 0.09*

All values are mean ± SEM, n=6 , * p<0.05 vs. control

In the Figure 2a, 2b the parameters showed that placentrex had the best wound healing result in excision wound healing model that may be comparable to the higher dose formulation, i.e. NSOINT-10. Low dose of volatile oil (NSOINT-5) had shown lesser activity compared to high dose but both the doses performed better than that of povidone iodine. From one way ANOVA analysis by post parametric Dunnet’s test it is evident that all the formulations performed significantly better than the control at 95% significance level.

Histology of granulation tissues obtained from the animals of dead space wound model were stained with hematoxilin-eosin dye and observed

The breaking strength of 10 day’s old granulation tissue was significantly promoted by NS (250mg/kg) and NS (500mg/kg). The dry tissue weight and hydroxyproline content also significantly increased in NS (250mg/kg) and NS (500mg/kg) when compared with control group. (Table: 6) From the above findings and the histology slides it was evident that Nigella sativa promotes wound healing by hastening the healing mechanism and promoting the formation of wound collagen.

Skin irritation study:

Low dose as well as high dose of volatile oil of Nigella sativa in PEG ointment used for skin irritation study, both the formulation as well as the PEG ointment base did not show any type of irritation. There was no evidence of showing any noticeable inflammation (Table: 7)

Table 7: Skin irritation study of Volatile oil of Nigella sativa

Group	Primary Irritation Score
PEG oint base (Control)	0
Ns (5%) in PEG oint base	0
Ns (10%) in PEG oint base	0

CONCLUSION:

The present study was undertaken to evaluate whether the volatile oil of Nigella sativa seed could promote wound healing in experimentally produced wounds in rats. The observations substantiate the use of Nigella sativa seeds in folklore medicine for the treatment of wounds. The study was designed to extract the volatile oil from the Nigella sativa seed by steam distillation method. The oil thus separated was used in a formulation by using PEG as ointment base, which was used for in vivo wound healing study on rats.

The formulation applied topically or given orally promoted the breaking strength, wound contraction and period of epithelialization. Collagenation, wound contraction and epithelization are crucial phases of wound healing. The preliminary phases of inflammation, fibroplasias, macrophasia, and collagenation are all interconnected. Thus if any one of these phase is intervened by drugs it may lead to either promotion or depression of the collagenation phase of healing process.

Growth hormone promotes the healing process by enhancing epithelial cell proliferation and cell collagen formation. Collagen is the main structural component of tissues such as cartilages and fibrous tissues. The collagen synthesis is promoted by different growth factors. Growth hormone promotes the proliferation of fibroblasts, and it in turn forms the granulation tissue. In the dead space wound model, Nigella sativa treatment was found to increase the granuloma tissue weight as well as breaking strength. Therefore, it can be assumed that the pro-healing activity of Nigella sativa could be due to the direct or indirect influence on growth hormone related activity.

Lipid peroxidation is an important process found in injuries like burn, skin ulcers and inflicted wounds. Drugs inhibiting lipid peroxidation may increase the stability of collagen fibrils, enhances the strength of collagen fibers, thereby preventing cell damage.

It is reported that antioxidants, such as metronidazole, vitaminC, vitaminE have been shown to promote wound contraction and epithelialization.¹³ The antioxidant property of the Nigella sativa, may be responsible for prohealing the action of wound healing.

Since the formulation was applied topically there may be chances of skin irritation. The skin irritation study on the rabbit skin proved that the volatile oil of Nigella sativa does not produce any significant irritation or inflammation on the skin.

Since volatile oil of Nigella sativa seed extracted by steam distillation produced wound healing effect compared to that of standard drug Placentrex in excision and incision wounds. It may lead to development of a wound healing agent that may turn out to be a promising agent in not only open wounds but also leg ulcer, skin grafting and severe types of burn and diabetic wounds.

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3. Zaoui A, Cherrah Y, Lacaille-Dubois MA, Settaf A, Amarouch H and Hassar M. Diuretic and hypotensive effects of Nigella sativa in the spontaneously hypertensive rat. Thérapie. 55(3); 2000: 379-382.

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5. Al-Ghamdi M. The anti-inflammatory, analgesic and antipyretic activity of Nigella sativa. Journal of ethnopharmacology. 76; 2001: 45-48.

6. Worthen DR, Ghosheh OA and Crooks PA. The in vitro anti-tumor activity of some crude and purified components of blackseed, Nigella sativa L. Anticancer research. 18(3A); 1998: 1527-1532.

7. Khan AW, Kotta S, Ansari SH, Sharma RK, Kumar A and Ali J. Formulation development, optimization and evaluation of Aloe vera gel for wound healing. Pharmacognosy magazine. 9(Suppl 1); 2013: S6-S10.

8. El-Fatatry H. Isolation and structure assignment of an antimicrobial principle from the volatile oil of Nigella sativa L. seeds. Pharmazie. 30(2); 1975: 109-111.

9. Burits M and Bucar F. Antioxidant activity of Nigella sativa essential oil. Phytotherapy research. 14(5); 2000: 323-328.

10. Midwood KS, Williams LV and Schwarzbauer JE. Tissue repair and the dynamics of the extracellular matrix. The International Journal of Biochemistry Cell biology. 36(6); 2004: 1031-1037.

11. Mansour M and Tornhamre S. Inhibition of 5-lipoxygenase and leukotriene C4 synthase in human blood cells by thymoquinone. Journal of Enzyme Inhibition and Medicinal Chemistry. 19(5); 2004: 431-436.

12. Senel O, Cetinkale O, Ozbay G, Ahçioğlu F and Bulan R. Oxygen free radicals impair wound healing in ischemic rat skin. Annals of Plastic Surgery. 39(5); 1997: 516-523.

13. Rao C and Ghosh A. Does metronidazole reduce lipid peroxidation in burn injuries? Indian Journal of Pharmacology. 29; 1975: 29-32.

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16. Hajhashemi V, Ghannadi A and Jafarabadi H. Black cumin seed essential oil, as a potent analgesic and antiinflammatory drug. Phytotherapy research. 18(3); 2004: 195-199.

17. Norwood AA, Tucci M and Benghuzzi H. A comparison of 5-fluorouracil and natural chemotherapeutic agents, EGCG and thymoquinone, delivered by sustained drug delivery on colon cancer cells. Biomedical Sciences Instrumentation. 43; 2007: 272-277.

18. The Ayurvedic Pharmacopoeia of India Part-I. Vol. I. Department of Ayus, Ministry of Health and Family Welfare, Government of India; 2008. p. 157.

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Received on 27.07.2016 Modified on 09.10.2016

Research J. Pharm. and Tech 2016; 9(12):2097-2105.

DOI: 10.5958/0974-360X.2016.00427.3

Parameter studied	Breaking strength (gms)
Control (PEG oint)	290.00 ± 6.19
Placentrax gel	492.50 ± 5.28*
Povidone Iodine	410.00 ± 6.95*
NSOINT-5	457.50 ± 3.82*
NSOINT-10	510.00 ± 5.32*

Parameter studied	Breaking strength (gm)	Dry tissue weight (mg)	Concentration of hydroxyproline (mg/ml)
Control	380.00+ 5.63	91.55 + 1.29	2966.66 + 80.27
N S 250mg/kg rbw	556.66+ 8.03*	172.35 + 4.38*	6500.00 + 73.03*
N S 500mg/kg rbw	611.66+ 6.28*	196.45 + 2.75*	7816.66 + 186.93*

Group	Observation	Inference
Control	Very less collagen formed. More number of mononeuclear inflammatory cell(macrophages) were present	Healing is not completed
N S 250mg/kg rbw	Increased collagenation, very few macrophages and capillaries were present	Healing process nearly completed
N S 500mg/kg rbw	Well formed collagen tissue and nearly absence of any inflammatory cell	Healing completed

MATERIALS AND METHODS:

Animals

Chemicals:

Extraction of volatile oil from Nigella sativa seeds:

Preparation of Topical ointment:

Acute oral toxicity study:

Excision wound model:

Incision wound model:

Dead space wound model:²⁴

Hydroxyproline Determination:

Histology of healed wound tissue:

Skin irritation study:

RESULTS AND DISCUSSION:

Acute oral toxicity study in rats:

Study of excision wound model with PEG ointment:

Study of incision wound model with PEG ointment:

Study of Dead space wound model:

Skin irritation study:

CONCLUSION:

REFERENCES:

MATERIALS AND METHODS:

Animals

Chemicals:

Extraction of volatile oil from Nigella sativa seeds:

Preparation of Topical ointment:

Acute oral toxicity study:

Excision wound model:

Incision wound model:

Dead space wound model:24

Hydroxyproline Determination:

Histology of healed wound tissue:

Skin irritation study:

RESULTS AND DISCUSSION:

Acute oral toxicity study in rats:

Study of excision wound model with PEG ointment:

Study of incision wound model with PEG ointment:

Study of Dead space wound model:

Skin irritation study:

CONCLUSION:

REFERENCES:

Dead space wound model:²⁴