Efficacy of Jasminum grandiflorum L. leaf extract on dermal wound healing in rats

Abstract

Wound healing is a fundamental response to tissue injury and natural products accelerate the healing process. Here, we have explored the efficacy of topical administration of an ointment, prepared by methanolic extract of Jasminum grandiflorum L. (Oleaceae) leaves, on cutaneous wound healing in rats. The topical application of the Jasminum ointment on full thickness excision wounds accelerated the healing process. Tissue growth and collagen synthesis were significantly higher determined by total hydroxyl proline, hexosamine, protein and DNA content. The response was concentration‐ and time‐dependent, when observed on days 4, 8 and 12 after wound creation. The rate of wound healing was faster as determined by wound contraction, tensile strength and other histopathological changes. In addition, this ointment also raised the activity of superoxide dismutase (SOD) and catalase (CAT) with high GSH content and low lipid peroxidation products in wound tissue. Thus, it could be suggested that the ointment from the methanolic extract of J. grandiflorum leaf improves the rate of wound healing by enhancing the rate of collagen synthesis and also by improving the antioxidant status in the newly synthesised healing wound tissue.

Introduction

The use of herbal medicines is on a continuous rise for health care. About 60% of the world population depends on traditional medicine for their primary health care (1) with emphasis on wound healing (2) and metabolic disorders. These medicines have fewer side effects as compared with conventional medicine. In wound healing, they act through multiple pathways to prevent inflammation and oxidative stress. They also act for disinfection, debridement and high collagen synthesis (3).

Jasminum grandiflorum L. (Oleaceae), commonly known as ‘Chameli’ (Hindi), is a glabrous twining shrub. In Ayurvedic texts, its leaves and flowers are used in the treatment of odontalgia, fixing loose teeth, ulcerative stomatitis, leprosy, skin diseases, ottorrhoea, otalgia, stangury, dysmenorrhoea, ulcers, wounds and corns. The Jati Gitra is one of its common formulations described in Ayurveda for management of wound healing (4).

Earlier reports have shown its antioxidant and anti‐ulcer (5), chemopreventive, anti‐lipid peroxidative (6), spasmolytic (7) and angiotensin converting enzyme (ACE) inhibitor property (8). It is rich in iridoid‐type compounds, secoiridoid glucoside named oleuropein, triterpenes, flavonoids, lignans and alkaloids ^{9, 10, 11}.

Wound healing is a complex multifactorial process, resulting in contraction and closure of the wound and restoration of a functional barrier (12). It includes inflammation, proliferation and migration of different cell types in a sequential manner (13). It involves two basic steps: (i) removal of damaged tissues and (ii) restoration of cutaneous or visceral architecture ^{14, 15}. Cutaneous wound healing primarily involves coordinated interaction between a number of cell types, extracellular matrix (ECM) molecules and growth factors in three overlapping phases: (i) coagulation and inflammation, (ii) proliferation and (iii) remodelling ^{16, 17}. Inflammation is part of the acute response of wound healing which attracts neutrophils at the wound site.

Delay in wound healing may be due to infection and high oxidative stress, resulting in a prolonged inflammatory step. The white blood cells accumulated at the wound site produce free radicals through a ‘respiratory burst' (18). Wound‐related non phagocytic cells also generate free radicals by involving non phagocytic NAD(P)H oxidase mechanism (19). Thus, the wound site is rich in both oxygen and nitrogen‐centred reactive species resulting in oxidative stress, high lipid peroxidation, DNA breakage and enzyme inactivation. The free radical scavenger enzymes, namely superoxide dismutase (SOD) and catalase (CAT) are also downregulated (20).

Thus, use of external antioxidants after the inflammatory phase would prove to be beneficial for wound healing (21). As antioxidant potential is high in natural products (22) because of their rich polyphenolic content ^{23, 24}, it is hypothesised that topical or systemic application of these herbal formulations would improve wound healing (25). Thus, here we have explored the effect of the polar methanolic extract of J. grandiflorum leaves on the rate of healing of cutaneous wounds by topical application. We have also investigated its role on the regulation of oxidative stress and collagen synthesis to understand its mechanisms.

Methods

Chemicals

L‐hydroxyproline, glucuronic acid, calf thymus DNA, chloramine‐T and bovine serum albumin were purchased from Sigma Chemical Co., St Louis, MO. p‐Dimethyl aminobenzaldehyde and Folin's Phenol reagent were arranged from Loba Chemie, Mumbai, India. Methyl cellosolve was obtained from E. Merck, Darmstadt, Germany. All other chemicals and reagents were of high analytical grade.

Plant extract preparation and standardisation

Leaves of J. grandiflorum L. were collected from the garden of our Institute and authenticated by pharmacognostical parameters by Professor KN Dwivedi from the Department of Dravyaguna, Faculty of Ayurveda, BHU. The voucher specimen of this sample was preserved in our department (voucher specimen no YBT/MC/14/1‐2008. The leaves were dried, powdered and its weighed amount was extracted with methanol in continuous soxhlet extraction apparatus for 24 hours. The solvent‐free dried extract was weighed to determine the percentage yield, which was 12%, w/w). The methanolic extract was subjected to qualitative tests for saponin (foam formation), flavonoids (using magnesium and dil HCl), phenolics (Folin–Ciocalteu assay), glycosides (Molisch's test), triterpenes and steroids (Liebermann–Burchard's test) by standard methods ^{26, 27}. It showed total phenolic content as 2·25 ± 0·105 [mg of galic acid equivalent (GAE)/g]. The results of preliminary phytochemical screening of the methanolic extract showed the presence of flavanoid, phenol, terpenoid, tennin and saponin. The extract was further standardised in terms of gas chromatography mass spectroscopy (GCMS) (28), which showed the presence of phytol and farnesol isomers as major compounds (data not shown).

Preparation and characterisation of ointment

The ointments were prepared with two different concentrations of the total methanolic extract of the J. grandiflorum leaves [2% and 4% (w/w)]. These concentrations were arrived at by initial experiments with different doses of the extract applied (data not shown). For ointment preparation, the extract was triturated with white soft paraffin base in a ceramic mortar and pestle and mortar (29). The ointment was standardised in terms of pH change in 60 days; viscosity, spreading capacity, skin irritation and rheological behaviours were assessed by the Rotational Brookfield viscometer (Malvern Instruments Ltd, Worcestershire, United Kingdom). The constituents of the ointment are described in Table 1.

Table 1.

Content of ointment prepared by methanolic extract of Jasminum grandiflorum leaves

S. no.	Constituents	Concentration
1	Total methanolic extract	2% or 4%
2	Light liquid paraffin	12%
3	Hard paraffin	3%
4	Cetosteryl alcohol	8%
5	Butylated hydroxy toluene	0·1%
7	Propylene glycol	5%
8	Methyl paraben	0·2%
9	Propyl paraben	0·02%
10	Glycerine	5%
11	Sodium lauryl sulphate	0·5%
12	Water	(Qs) sufficient quantity to make 100 ml

Wound preparation and grouping of animals

All experimental protocols were approved by ‘Ethical Committee for Animal Welfare’ of Institute of Medical Sciences (IMS). Male albino rats [Charles Foster (CF) strain] of 150–200 g body weight were purchased from the central animal house at the Institute and acclimatised in our laboratory conditions for 6 days in a 12‐hour light:12‐hour dark cycle with free access to water and food.

Two experiments were carried out. In one set, excision‐type wound was created and in the second set, incision‐type wound was made on the back of animals (30). In both the models the animals were randomly divided into three groups, of six animals in each. Animals in group I (experimental control): topical application of ointment base, group II: treated with 2% ointment and group III: treated with 4% ointment.

For wound preparation, animals were anaesthetised by an intraperitoneal injection of thiopentone (25 mg/kg). The dorsal surface of the rat was shaved and the underlying skin was cleaned with 70% ethanol. For excision‐type wound, a circular cut of 2 × 2 cm² was made by using a scalpel blade to the depth of loose subcutaneous tissues (31). Different types of ointments were applied twice daily for 12 days and finally the wound tissues were removed to analyse the biochemical parameter. The histopathological changes and antioxidant parameters were determined only on day 12. A separate group of animals was assessed for the effects of the drug and the degree of wound contraction on days 4, 8 and 12. Further, in the case of incision wound, a paravertebral cut of 6 cm length was made on the back of each animal in anaesthetised condition. The cut was 1 cm laterally away from the vertebral column, linear and deep. The wound was then closed with interrupted sutures 1 cm apart using a surgical thread and a curved needle. It was subjected to the ointment treatment protocol as described in the previous experiment. On day 10, tensile strength of wound skin was determined. For measurement, the sutures were removed on day 9, wounding and tensile strength was measured on day 10. Both sides of the cut were hooked; one side was fixed with the base and the other side was subjected to gradual addition of water from a vessel attached to the hook. The tensile strength was reported as the minimum weight (g) of water, necessary to break the united skin. The breaking of the healed cut was visually observed.

Rate of wound contraction

The rate of wound contraction was expressed as percentage contraction of the area of open wound, as recorded on transparent paper (32), by using following formula to calculate the percentage of wound contraction:

(1)

Estimation of protein and DNA content

Tissue (100 mg wet wt.) was mixed with 10 ml of 5% Trichloro acetic acid (TCA) solution and incubated at 90°C for 30 minutes in a water bath to extract protein and DNA (33). The solution was centrifuged and the supernatant liquid was used to estimate DNA by the method of Burton (34) and protein by the method of Lowry (35).

For estimation of hydroxyproline and hexosamine, the granulation tissue was dried in a hot air oven at 70°C until it attained constant weight. The acid hydrolysate of the dry tissue was used to estimate hydroxyproline as defined by Woessner (36) and hexosamine by Elson method (37). These values were expressed as mg/g dry weight of the tissue.

Histopathological changes

The isolated tissues were fixed in 10% formalin. It was dehydrated through graded alcohol series, cleared in xylene and embedded in paraffin wax (melting point, 56°C). Serial sections of 5 µm were cut and stained with haematoxylin and eosin (H&E) stain. The sections were examined under a light microscope and photgraphed.

Analysis of antioxidant‐related parameters

The excised granulation tissue was rinsed with ice‐cold normal saline (0·9% NaCl) containing 0·16 mg/ml heparin and homogenised in 0·1% phosphate buffer (pH 7) containing protease inhibitor in Reflon homogenizer to get 10% homogenate (w/v). Reduced glutathione (GSH) level was measured by colorimetric method as protein‐free sulfhydryl content, by using 5,5‐dithiobis‐2‐nitrobenzoic acid (DTNB) ^{38, 39}. Here, 0·5 ml of homogenate was diluted with equal volume of phosphate buffer (0·2 M) and then 2 ml of DTNB reagent (1 M) was added. The developed color read at 412 nm against a blank containing 5% TCA instead of homogenate. SOD activity was assessed in terms of its ability to inhibit the reduction of nitro blue tetrazolium (NBT) by superoxide radicals ^{40, 41}. CAT activity was determined on the basis of the rate of decomposition of H₂O₂ by monitoring the decrease in absorbance at 249 nm (1983). The level of lipid peroxides was measured as thiobarbituric acid reactive substance (TBARS). 1′1′3′3′‐tetramethoxypropane was used as standard ^{42, 43}. Total protein content was determined by Lowry's method using bovine serum albumin as a standard (35).

Statistical analysis

The data are expressed as mean ± SD and statistical significance between experimental and control groups were analysed by one‐way ANOVA followed by post hoc Dunnet test. P values <0·05 were considered statistically significant and P values <0·01 were considered as highly significant. All statistical analyses were performed using SPSS statistical version 16·0 software package (SPSS R Inc., Chicago, IL).

Results

Evaluation of physical parameters of ointment

There was slight increase in pH, up to 30 days, but it was within the physiological range of human skin, i.e. pH 5–pH 6. Both formulations did not produce any skin irritation, that is, erythaema and oedema, during observation for 12 days. A comparative study of viscosity and spreading ability showed inverse relation (Table 2).

Table 2.

Physical evaluation of ointment

Time period (days)	Ointment formulation (%)	pH	Viscosity (cps)	Spreadibility	Homogeneity
0	2	6·3 ± 0·18	15·98 ± 0·08	27·89 ± 1·45	Pass
	4	6·5 ± 0·23	16·45 ± 0·05	27·12 ± 1·34	Pass
15	2	6·2 ± 0·12	16·12 ± 0·08	26·25 ± 1·45	Pass
	4	6·47 ± 0·20	17·14 ± 0·06	25·92 ± 1·02	Pass
30	2	6·1 ± 0·14	17·24 ± 0·04	25·65 ± 1·45	Pass
	4	6·2 ± 0·12	17·93 ± 0·08	24·34 ± 1·24	Pass
60	2	7·4 ± 0·21	18·45 ± 0·06	23·01 ± 1·26	Pass
	4	7·3 ± 0·24	18·64 ± 0·07	22·56 ± 1·34	Pass

Rate of wound contraction

The rate of wound contraction was significantly high in ointment‐treated wounds. The response was concentration‐ and time‐dependent. A 2% of ointment extract produced 76·35% contraction and 4% extract produced 96·12% contraction on day 12 (Table 3); the changes were statistically significant (P ^** < 0·01) when compared to normal control group where it was only 62·94 ± 0·35% in comparison with total wound closure. The ointment application reduced this period to 14 days which was 6 days lesser than the normal control group (Figure 1).

Table 3.

Effects of different concentration of ointment of Jasminum grandiflorum leave's methanolic extract ointment on rate of wound closure

Groups	% of wound contraction on different days			Number of days required for complete wound closure
	4th	8th	12th
Normal control	25·66 ± 0·64	48·75 ± 0·54	62·94 ± 0·35	20 ± 0·31
2% J. grandiflorum ointment in normal wound	35·01 ± 1·56	58·60 ± 2·98 *	76·35 ± 1·46 *	18 ± 1·46
4% J. grandiflorum ointment in normal wound	40·68 ± 1·86 **	70·19 ± 3·82 **	96·16 ± 2·42 **	14 ± 2·42

^aValues were significant (P < 0·05);

^bvalues are highly significant (P < 0·01) when compared with diabetic control value.

Figure 1.

Effects of different concentration and different duration of Jasminum grandiflorum methanolic extract ointment treatment on rate of wound healing (expressed in terms of the size of the wound area).

Tensile strength of incision wound

Tensile strength of healing tissues was significantly higher by 4% ointment‐treated wounds on day 10 as compared with the untreated wound (P ^* < 0·01). The response was dose‐dependent (Figure 2).

Figure 2.

Effects of Jasminum grandiflorum leaves of methanolic extract ointment treatment on tensile strength of wound healing tissue in normal incision wound model in rats.

Hydroxyproline and hexosamine content

The content of hydroxyproline and hexosamine in the granulation tissues were significantly higher in the ointment‐treated group as compared with control rats. It was dose and time‐dependent (Table 4) compared to days 8 and 12. On day 12, the hydroxyproline content increased by 2·28‐fold in the 4% ointment‐treated group when compared with control group. It was about 1·94‐fold higher in case of hexosamine. The rise in hydroxyproline content is considered the marker of collagen synthesis, which not only confers strength and integrity to the tissue matrix but also plays an important role in haemostasis and in epithelisation phase of wound healing (44). The increase in hexosamine of in the granulation tissue provides strength to the regenerated tissue in normal wound.

Table 4.

Effects of Jasminum grandiflorum leaves methanolic extract ointment on various biochemical parameters in granulation tissue

	4th day	8th day	12th day
Hydroxyproline (mg/g wt)
Normal control	22·67 ± 1·11	30·95 ± 2·30	36·85 ± 2·98
2% J. grandiflorum ointment in normal wound	35·30 ± 1·42 *	46·08 ± 2·92 *	61·26 ± 2·50 **
4% J. grandiflorum ointment in normal wound	48·67 ± 3·71 **	69·05 ± 5·58 **	84·03 ± 2·36 **
Hexosamine (mg/g tissue wt)
Normal control	3·58 ± 0·25	3·68 ± 0·14	4·40 ± 0·14
2% J. grandiflorum ointment in normal wound	4·83 ± 0·678	5·09 ± 0·24 *	6·45 ± 0·29 *
4% J. grandiflorum ointment in normal wound	6·42 ± 0·77 *	7·16 ± 0·68 **	8·56 ± 0·42 **
DNA(mg/g wet tissue wt)
Normal control	1·73 ± 0·097	4·50 ± 0·81	4·96 ± 0·21
2% J. grandiflorum ointment in normal wound	2·15 ± 0·058	6·01 ± 0·15 *	6·67 ± 0·19 *
4% J. grandiflorum ointment in normal wound	3·12 ± 0·197 **	8·65 ± 0·18 **	9·98 ± 0·21 **
Protein (mg/g wet tissue wt)
Normal control	40·59 ± 1·29	60·45 ± 1·74	61·12 ± 2·04
2% J. grandiflorum ointment in normal wound	58·79 ± 1·25 *	89·24 ± 5·73 *	96·31 ± 2·04 *
4% J. grandiflorum ointment in normal wound	68·10 ± 0·374 *	105·65 ± 5·42 **	117·21 ± 3·04 **

Values were significant ( ^* P < 0·05); ^**values are highly significant (^** P < 0·01) when compared with diabetic control.

Protein and DNA content

There was a significant increase in the DNA and protein content in the ointment‐treated group as compared with normal control animals. On day 12 of the 4% ointment‐treated group, the DNA content was enhanced 2·01‐fold and the protein content was raised 1·91‐fold as compared with the control group (Table 4).

Histopathological evaluation

Histology of the wound tissue of the control animals showed the presence of more inflammatory cells, less fibroblast cells and collagen content, fewer new blood vessels as compared with the drug‐treated group (Figure 3). However, the tensile strength of the granulation tissue of the treated group showed higher collagen and fibros tissue with a higher number of blood vessels in a concentration‐dependent manner.

Figure 3.

Effect of topical application of ointment of methanolic extract of Jasminum grandiflorum leaves (2% and 4%) for 12 days on histological changes in granulation tissue [haematoxylin and eosin (H&E) stained]. H&E stained histological section of cutaneous wound site obtained from controls and J. grandiflorum ointment‐treated rats on day 12 after wounding. (A) Untreated wound, showing more inflammatory cells and incomplete epithelisation. (B) Treated wound with 2% ointment showing moderate inflammatory cells and increase the epithelisation and less neovascularisation. (C and D) Treated wound with 4% ointment exhibit more neovascularisation, less inflammatory cells and well organized thick epithelisation. (E) Thick and immature epidermis; (arrow) inflammatory cells and (arrow head) neovascularisation, 100×.

Antioxidant status

The results showed high GSH (45·99%) content in the ointment‐treated tissue. This was also supported by lower lipid peroxidation, which was measured as the amount of TBARS. The ointment‐treated tissue showed high activity of SOD and CAT enzymes when compared with normal untreated sham‐control animals. High protein and DNA content/unit wt of tissue supports more tissue synthesis. All these changes were concentration‐dependent (Table 5).

Table 5.

Effect of 12 days treatment with Jasminum grandiflorum leave's methanolic extract‐ointment on lipid peroxide and antioxidant enzymes in wound granulation tissue

Parameter	Normal control	2% J. grandiflorum ointment in normal wound	4% J. grandiflorum ointment in normal wound
TBARS (nmol/mg protein)	1·37 ± 0·083	1·06 ± 0·035	0·930 ± 0·021 *
GSH (µg/mg protein)	1·55 ± 0·062	2·11 ± 0·06	2·87 ± 0·12 *
Catalase (U/mg protein)	5·87 ± 0·45	7·05 ± 0·61 *	8·64 ± 0·63 **
Superoxide dismutase (U/mg protein)	2·64 ± 0·13	2·96 ± 0·15	3·84 ± 0·19 *

GSH, glutathione; TBAR, thiobarbituric acid reacting substance. Values are mean ± SD; n = 6.

^*Values were significant (P < 0·05);

^**values are highly significant (P < 0·01) when compared with diabetic control value.

Discussion and conclusion

Wound healing is the physiological response to the tissue injury that restores the integrity of damaged tissues. Its mechanistic part comprises of four basic steps, e.g. inflammation, wound contraction, epithelialisation and granulation tissue formation. Inflammation starts immediately after the disruption of tissue integrity. The platelets adhered to the wound surface along with clotting factors, and forms a haemostatic plug to stop bleeding. The prostaglandins (PGE1 and PGE2), the primary cause of acute inflammation, are released at this stage. Further, the active motile white blood cells migrate to the wound area engulfing cellular debris. In the initial stages, wound contraction begins slowly, which enhances after 3–4 days resulting in the appearance of myofibroblsts at the wound's margin.

The incision wound model has been used to determine the tensile strength, which is proportional to the degree of collagen formation (45). However, the excision wound model helps in assessment of rate of wound healing with respect to time. Both the models have shown positive and dose‐dependent effects from the methanolic ointment extract of J. grandiflorum. Its enhanced cellular proliferation and collagen synthesis, evidenced by increased hydroxyproline content of granulation tissues is an important constituent of ECM. Increased hexosamine content reflects the stabilisation of collagen molecules by enhancing electrostatic and ionic interactions (46). Collagen not only confers strength and integrity to the tissue matrix but also plays an important role in homeostasis and in epithelialisation at the latter phase of healing (44). Hence, enhanced synthesis of hydroxyproline and hexosamine in treated rats provides strength to the repaired tissue and also healing pattern. Increased protein and DNA content per unit weight of granulation tissues indicate high degree of tissue formation under the effect of ointment treatment.

Our biochemical findings correlate very well with the histological findings. A close examination of granulation tissue sections revealed that tissue regeneration was much faster in the treated group compared to wounds in the control group. There was increased blood vessel formation (47) and enhanced collagen fibre in the treated group. The presence of myofibroblasts is considered to be characteristic of tissue undergoing contraction ^{48, 49}.

An imbalance between the oxidant and antioxidant defense mechanism leads to oxidative stress. It results in lipid peroxidation, DNA damage and enzyme inactivation responsible for scavenging free radicals (20). Further, oxygen‐free radicals or oxidants contribute to tissue damage in the events following skin injury and are known to create impairment in the healing process (50). Antioxidants, on the other hand, significantly prevent tissue damage and stimulate the wound‐healing process (51).

Reactive oxygen species (ROS), although vital to wound healing in early stage and in lower concentration, has a pathological role, if persisted for longer period (50). It promotes vascular endothelial growth factor (VEGF) expressions in keratinocytes (52) in homeostatic condition. The spontaneously generated superoxide, because of respiratory burst (18), may get converted to H₂O₂, which produces highly reactive OH radicals, resulting in the inhibition of proliferation and also apoptosis of newly generated cells. This is evident by low DNA and protein content in experimental control group. Their higher content in J. grandiflorum ointment‐treated wound indicates that the high rate of collagen‐tissue synthesis (50).

This free radical scavenging property may be due to direct action of phenolic compounds of the J. grandiflorum ointment or because of synthesis of antioxidant enzymes. Interestingly, our results clearly indicate the increased activity of SOD and CAT, which supports its indirect antioxidant property through activation of enzymes. Also, these phenolics act either through their redox action or through metal chelation properties (53). We have already reported similar antioxidant and metal chelation property along with net anti‐inflammatory potential of J. grandiflorum leaves (28). Thus, this observed rapid wound healing with J. grandiflorum ointment could be attributed to these reported properties of J. grandiflorum leaves.

Topically administered drugs are more effective in wound contraction, probably, because of its larger availability at the wound site. Recent studies with other plant extracts have also shown that phytochemical constituents like flavanoids (54), triterpenoids (55) and tannins (56) are known to promote the wound‐healing process. The phytochemical screening of methanolic extract of J. grandiflorum showed the presence of secoiridoid glucoside, triterpenes, flavonoids, lignans, ^{9, 10} and other phenolic compounds (57), which supported its antioxidant mediated wound‐healing process.

Thus, it could be concluded that topical use of ointment, made up of the methanolic extract of J. grandiflorum leaves is beneficial in the management of acute wound. Its mechanism of action might be through its free radical scavenging property.

Acknowledgements

The authors are thankful to the Council of Scientific and Industrial Research and the Board of Research in Nuclear Sciences for financial assistance to conduct this study. The authors also express their thanks to the Bananas Hindu University for providing scholarship to associated student. The authors extend their thanks to Prof. VK Shukla, Department of Surgery, IMS, BHU for valuable discussion.