Preferential inhibition of bacterial elastase over human neutrophil elastase by leaf extracts of Psidium guajava: an in vitro study.
Since the advent of civilization, plants have been used as a source of medicine. Recently, there has been a renewed interest in plant extracts that have medicinal properties, especially as these have been found useful in identifying molecules of importance in the pharmaceutical industry. Psidium guajava is cultivated commercially for its fruit, but its leaves, bark, and roots have a long history of medicinal use. The leaves of the guava plant are in use to treat wounds and ulcers, and an infusion of the leaves has been used to treat rheumatism, epilepsy, chorea, and diarrhea. [1,2] Phytochemical studies on P. guajava have shown the presence of tannins, triterpenes, flavonoids, and phenolic compounds [3-5] Tannins present in the plant have been shown to act against bacteria by inactivating microbial adhesions and enzymes.  The growth of microorganisms can also be inhibited by essential oils in leaves.  Besides these, some plants also have antimicrobial proteins that play a role in defensive mechanism against bacteria, fungi, and viruses. Plants synthesize inhibitory proteins that suppress the extracellular enzyme activities released by pathogenic microorganisms [8,9] These protease inhibitors (PIs) are of common occurrence and some serine PIs (serpins) involved in inhibiting proteases of plant pathogens have been isolated and used for medical treatments. [10-12] The observations on the antibacterial activity of P. guajava is nonspecific. Most studies have shown that there was a zone of inhibition when the leaf extract was applied on the microbial culture plate.  The mechanism as to whether its antimicrobial activity  is by inhibition of bacterial proteases of different origin is not clear. Elastase released by microorganisms is an important virulence factor in several types of infections,  and the invasive effect of elastase by Pseudomonas aeruginosa has been well documented.  Human neutrophil elastase (HNE) is involved in the degradation of bacteria and immune complexes phagocytosed by polymorphonuclear leucocytes.  As these leaf extracts are used to facilitate wound healing, it was felt appropriate to assess the inhibitory abilities of the P. guajava leaf extract on HNE and P. aeruginosa elastase to test the specificity of their inhibition on these enzymes. This study also explored the presence of PIs of protein nature present in the leaf extract.
MATERIALS AND METHODS
All chemicals used for the study were of analytical grade unless otherwise mentioned. Succinyl trialanyl p-nitroanilide (STANA) and HNE were obtained from Sigma, USA, and tannic acid used as standard was from Merck.
Cultivation and Growth of the Microorganism
The microorganism P. aeruginosa MTCC 3541 was purchased in a lyophilized form from Microbial Type Culture Collection and Gene Bank, Chandigarh, India. It was rehydrated and cultivated on nutrient agar plates for 24 h at 37[degrees]C. Protease release by the microorganism was confirmed by noting zone of clearance on caseinate agar plates, which were streaked and kept for incubation at 37[degrees]C for 48 h. The strain was then inoculated into a nutrient broth containing 1% glucose, 0.5% casein, 0.5% yeast extract, 0.2% [K.sub.2]HP[O.sub.4], 0.2% [K.sub.2]HP[O.sub.4], and 0.1% MgS[O.sub.4] 7[H.sub.2]O.  The broth was kept in a rotatory shaker at 120 rpm at 37[degrees]C for 24 h. It was then centrifuged at 10,000 rpm for 20 min at 4[degrees]C.
Purification of the Enzyme
The culture broth was subjected to 70% saturation with ammonium sulfate at 4[degrees]C in a cold room with constant stirring and allowed to stand overnight. It was then centrifuged at 10,000 rpm for 20 min. The precipitate was collected and dissolved in 200 mM Tris-HCl buffer, pH 8.0, and again centrifuged. The supernatant was collected and dialyzed against 200 mM Tris-HCl buffer, pH 8.0, with a change of buffers at every 12th h of dialysis. The dialysate (ammonium sulfate fraction) was collected and measured for protein by Lowry's method.  The elastase activity in this was determined using STANA as a substrate by the method of Bieth et al.  The fraction after ammonium sulfate precipitation was then subjected to further purification by ion exchange chromatography using DEAE cellulose column, which was equilibrated with 200 mM Tris HCl buffer. Elution was done in a stepwise manner using NaCl (0.05-0.5 M) in the same buffer. The eluted fraction that showed the highest proteolytic activity was then used as the source of the enzyme in this study. The protein content was estimated as per the method of Lowry and elastase activity as per Bieth et al. 
Preparation of the Leaf Extract
Plant Materials: The fresh mature leave of P. guajava were collected in Kolar District, Karnataka, in the month of August. The leaves were washed thoroughly with distilled water and dried at room temperature. The dried leaves were then ground uniformly in a mechanical grinder to give a fine powder.
Five grams of the powder was then mixed with 30 mLof methanol in an Erlenmeyer flask. It was kept in a shaking incubator at 120 rpm for 24 h. The mixture was the filtered using Whatman filter paper No. 1, dried, and stored at 4[degrees]C. It was then dissolved when required for use.
The aqueous extract was prepared similarly using hot (60[degrees]C) deionized water. After filtration, one part of the aqueous extract was washed with an equal volume of 70% cold acetone and kept overnight at 4[degrees]C. It was centrifuged at 3000 rpm, and the precipitate formed was removed, dissolved in 0.2 M Tris HCl buffer, pH 8.0, and again centrifuged at 3000 rpm. The process was repeated several times until the precipitate was colorless. The supernatant obtained was used as the source of aqueous extract without phenolic compounds. Protein content in this extract was measured by Lowry's method. 
The total tannin content of the leaf extract was determined by the Folin-Ciocalteu reagent method and compared with known standards prepared with tannic acid. The results obtained were expressed as micrograms of tannic acid per milliliter of the extract as per the method of Makkar. 
Two sets of assay systems were run. In one set, the source of the enzyme elastase was that obtained from P. aeruginosa by the process mentioned earlier. Similarly, a fixed volume of HNE was then used as the enzyme source in a second system. The concentration of both enzymes, i.e., HNE and the bacterial elastase, was fixed at 10 U/L to get consistent enzyme activity so that it hydrolyzed the substrate to give an optical activity of 0.25. The assay system comprised 200 mM of STANA in 200 mM of Tris HCl buffer, pH 8.0, and the reaction was initiated by the addition of the enzyme. After 15 min of incubation at 37[degrees]C, the reaction was stopped by the addition of 1 mL of 30% acetic acid, and the optical density of p-nitroaniline liberated was measured at 410 nm. One enzyme unit was defined as the amount of enzyme required to release 1 [micro]mol of p-nitroaniline per unit time (min).
Elastase Inhibition assay
The inhibitor sources in each set were varied concentrations of the methanolic and aqueous extracts of the leaves and the aqueous fraction (without phenols). All of them were in the assay buffer, i.e., 200 mM Tris HCl buffer, pH 8.0. The leaf extracts were preincubated for 10 min with a fixed volume of the enzyme. The reaction was initiated with a fixed volume of the substrate, i.e., 200 mM STANA. After 15 min incubation at 37[degrees]C, the reaction was stopped by the addition of 1 mL of 30% acetic acid, and the residual activity was determined by measuring optical density of p-nitroaniline liberated at 410 nm. Suitable controls were also run without the inhibitor. The inhibitory activity was determined by the difference between activity of the bacterial elastase without inhibitor and the residual activity of the same solution after adding the inhibitor. Similarly, the inhibitory activity on HNE was also determined. The percentage of inhibition was calculated using inhibition (%) = [1 - (B/A)] x 100 where A is the activity of the enzymes without inhibitor and B is the activity in presence of inhibitor. One inhibitor unit was defined as the amount of inhibitor required to inhibit 50% of the corresponding enzyme activity per unit time. [IC.sub.50] was determined for both elastase of P. aeruginosa and HNE with varying concentrations of the inhibitor in the assay systems.
The methanolic extract of the leaves showed good inhibitory capacity, more than that of the aqueous extract, against both enzymes, elastase of P. aeruginosa and HNE [Figures 1 and 2]. However, [IC.sub.50] (concentration in [micro]g/mL at which the inhibition of the enzyme activity is 50%) for HNE was 40 and 44 [micro]g/mL for the methanolic and aqueous extracts while, for bacterial elastase, 24 and 30 [micro]g/mL, respectively. [K.sub.i] values were calculated for methanolic and aqueous fractions and were 18.3 and 20.2 [micro]g/mL for HNE and 10.4 and 13.0 [micro]g/mL for bacterial elastase, respectively.
The aqueous extract after removal of phenolic substances with acetone did not exhibit any recordable inhibitory activity on both HNE and elastase of P. aeruginosa.
[FIGURE 1 OMITTED]
The guava leaves known for its antibacterial properties has not been explored for the mechanisms as to how the leaf extract exert its action on the microorganisms. This study emphasizes on the inhibitory role of the extract on elastase as it is shown as a virulence factor and plays an important role in infection. The inhibitor effect of the methanolic and aqueous extracts here showed that both elastase of P. aeruginosa and HNE are inhibited. The data analysis showed that the amount of inhibitor required for inhibition of P. aeruginosa is about 40% lower than that of HNE. The lower [K.sub.i] values for bacterial elastase in both methanolic and aqueous fractions suggest that it is more prone to the effect of the inhibitor in the leaves as the lower [K.sub.i] indicates that less amounts of the inhibitor brings about greater degree of inhibition. The strain of P. aeruginosa MTCC 3541 used in this study had elastase activity. We had cultivated the organism and determined its proteolytic activity on casein substrate (results not shown in this study) and determined the purified elastase activity on STANA.
An earlier study had shown that porcine pancreatic elastase is inhibited by methanolic leaf extract of P. guajava and that the triterpene derivatives present in the leaf brought about the elastase inhibition.  There have been studies on elastase inhibition by extracts of plants such as Areca catechu, Cornus kousa, and Achillea millefolium. But the inhibition was brought about by phenolic substances, flavonoids and triterpenoids. [23-25]
So, in this study, we wanted to establish whether the wound healing capacity of the leaf extract is owing to a PI present in the leaves whose inhibition would be specific or if inhibition of elastase was nonspecific. These PIs are able to selectively abrogate protease actions by forming less active or fully inactive complexes with their cognate enzymes; so, they can be used as drugs by themselves or serve as a template for development of highly specific drugs.  Hence, an aqueous extract, a methanolic extract, and an aqueous extract after removal of phenolic compounds of the leaves were prepared as sources of inhibition. Elastase release by microorganisms is an important invasive factor. So, it was thought appropriate to use an extracellular enzyme released by an invasive microorganism as only porcine pancreatic elastase had been used in earlier elastase inhibition studies. Inhibition of HNE has been shown by some medicinal plants in Yemen in an earlier study.  In our study, the methanolic extract of the leaves showed good inhibitory capacity, more than that of the aqueous extract, against both elastase of P. aeruginosa and HNE. This was probably because methanol as a solvent had extracted many of the active compounds present in the leaf more than that of water; so, percentage of inhibition brought about was more in the methanolic fraction.
[FIGURE 2 OMITTED]
During wound healing, in the initial stages, some amount of proteolytic activity is required to break down the extracellular matrix and promote wound healing. This role is usually provided by HNE.  As per the results of this study, application of the leaf extract appears to preferably inhibit bacterial elastase over that of HNE. So, there does appear to be a scientific basis for the application of these leaves on a wound. Attempts to isolate an inhibitor of protein nature, which would have been more specific in its inhibition, however, failed as the aqueous extract, which was treated with acetone and had the major part of the bioactive compounds washed out, showed negligible inhibitory activity. Moreover, when protein content was measured in this fraction, it was present only in trace amounts, indicating that not much water-soluble proteins were present under the given set of conditions.
The results of the study clearly demonstrate more specific inhibition of elastase of P.aeruginosa in comparison with HNE by the leaf extract of P. guajava. It provides a scientific basis that this leaf extract facilitates the wound-healing process by curtailing progress of invasion by microorganisms. So, there is scope for further isolation and chemical characterization of the active molecules present, which inhibit elastase of P. aeruginosa and exhibit potential use for topical application. However, its action on other vital enzymes and physiological processes in host systems need to be evaluated.
Deena Mendez, AV Moideen Kutty, SR Prasad
Department of Biochemistry, Sri Devaraj Urs Medical College, Tamaka, Kolar,
Correspondence to: AV Moideen Kutty, E-mail: firstname.lastname@example.org
Received October 27, 2015. Accepted October 31, 2015
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Source of Support: Nil, Conflict of Interest: None declared.
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|Author:||Mendez, Deena; Kutty, A.V. Moideen; Prasad, S.R.|
|Publication:||National Journal of Physiology, Pharmacy and Pharmacology|
|Date:||Mar 1, 2016|
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