MMP-2 Inhibitory Activity of PBS Extract of Guava Leaves

PBS (Phosphate Buffed Saline), pH 7.4 extract of Guava leaves (Psidium guajava L.) inhibits the active MMP-2 like molecule both of plant origin as well as active MMP-2 of human (breast cancer patients’ saliva) origin. The zymogram and ELISA were developed of different samples following standard methods. The isolation and identification of the new MMP-2 inhibitor(s) for its molecular characterization is under investigation.


Introduction
Ethnomedical searches for new bioactive molecules are becoming a promising field of science to discover a cure of different diseases. Traditionally, extract of different parts of guava (Psidium guajava L.) plants has been used from the ancient human history to treat different human illness. It is reported that guava leaves contain several phenolic compounds, which has shown to maintain a healthy blood glucose level efficiently [1]. Different world wild in vitro and in vivo research has demonstrated the potential pharmacological importance of leave extract can be a co-treatment for different physiological problems. Modern research acknowledges the importance of specific individual compounds from the bioactive guava leaves extract.
A study on human cancer reported that different compounds of guava leaves may have an anticancer activity and these compounds may inhibit the activation of AKT/mTOR/S6K1/MAPK pathway, but the detail mechanisms are not well understood yet [2]. The same study has also demonstrated a compound of guava leaves known as guava leaf hexane fraction (GHF) that can promote the production of different cytotoxic molecules and leads to cell death. GHF mediated apoptosis can be mediated by the inhibition of AKT/mTOR/S6K1/MAPK signaling pathway, but farther research needs to be done to understand the proper mechanisms [2]. These studies altogether propose that these molecules have the ability to suppress tumorigenesis by interfering several cell signaling pathways and thus it gives us a promising way to cure and prevent cancer.
We prepared a simple 1× PBS (pH 7.4) extract of Guava Leaves at 4˚C especially to see whether PBS extract of guava leaves has any effect on MMP-2 activity and observed that guava leaves PBS extract inhibits very efficiently the plant derived active MMP-2 like molecule [15] and also it inhibits salivary active MMP-2 of human breast cancer patients [16]. This study in future may be an important part in the management of cancer.

Methods & Materials
Guava Leaves was collected from RKMVERI's garden. The guava leaves was extracted using a Teflon homogenizer with cold 1× PBS (pH 7.4) in a proportion of 1:5 in ice at 4˚C for 2 hrs. The homogenate guava leaves was then centrifuged at 10,000 rpm at 4˚C for 30 mins. The clear supernatant was saved and kept at 4˚C as a source of the inhibitor of MMP-2. The zymography was run following the method of Bhattacharrya et al. [16]. The ELISA was run following the method of Mondal et al. [15]. The immunoblot was developed according to Mondal et al. [15]. The Gelatin was purchased from Sigma U.S.A. The monoclonal antibodies against MMP-2, MT1-MMP and TIMP-2 were purchased from Santa Cruz, USA.

ELISA
50 μg of the extract (sample) in triplicate was added/well of the ELISA plates.
Allow the plate with the samples to attach for one and half hour at 37˚C. The wells were then treated with 3% BSA for 1 hr. After washing the primary antibody in a dilution of 1:2000 (Santa Cruz, USA) were then added (50 μl) in triplicate and kept at 37˚C for one and a half hour. After three washing (PBS con-taining 0.1% Tween-20), second antibody coupled with alkaline phosphatase (50 μl, 1:2000 diluted) was added in triplicate for one and half hour at 37˚C. The TMB substrate was added. The stop solution were added after development of colour and read at 540 nm [15].

Immunoblot
To develop immunoblot 200 μg of salivary protein was run on 8% SDS-PAGE. The proteins were then transferred onto a nitrocellulose (NC) membrane electrically. The NC membrane was then blocked with 5% BSA for 2 hours at 37˚C in an incubator. Then after washing the membrane was reacted with MMP-2 primary monoclonal antibody (Santa Cruz, USA) for 1 and half hour followed by 2nd antibody coupled with alkaline phosphatase for the same time at 37˚C after washing (3 times). The colour was then developed with NBT/BCIP following Bhattacharyya et al. [16].    Figure 2(a) shows addition of guava leave extract in increasing concentration to Bamboo leaves extract inhibits the activity of MMP-2 like molecule extracted with PBS from Bamboo leaves [15]. Figure 2(b) also shows that in case of different MMP like molecule (not MMP-2 like molecule according to ELISA) is not inhibited by guava leaves extract. Figure 3(a) shows that other MMP-2 like molecule [15] in Nayantara flower's PBS extract [15] and newly found gandal leaves PBS extract. Figure 3(b) (not published) the MMP-2 like molecule is also being inhibited by Guava leaves extract. Figure 3(c) confirms that ELISA of Gandal leaves and Nasturtium flower extracts are MMP-2 positive with MMP-2 monoclonal antibody. We asked the question that whether this is possible for the MMP-2 inhibitor of Guava Leaves has any effect on human MMP-2. In 2017 we published [16] that human breast cancer female patients saliva has very high activated MMP-2 [16]. We collected saliva from breast cancer patients from R G Kar Medical College, Kolkata. Figure 4(a) shows that the comparative zymogram confirms that activated MMP-2 of human Saliva origin is being appreciably inhibited by Guava leaves extract (we repeated the experiment in several cases). Figure 4(b) shows that the PBS extract of Guava Leaves can inhibit efficiently (graded response) of the active MMP-2 of human (Breast cancer patients saliva). We suspected whether this increase in MMP-2 activity is due to MT1-MMP and also whether this inhibitor of MMP-2 has any similarity with the known MMP-2 inhibitor that is TIMP-2. Figure 5(a) shows that the ELISA of Guava leaves PBS extract has no TIMP-2 like activity. Perhaps the ELISA result indicates that MMP-inhibitor(s) present in PBS extract of Guava Leaves may be new type of inhibitor other than TIMP-2. Figure 5(b) ELISA test using human MT1-MMP monoclonal antibody shows that MT1-MMP is present in appreciable amount in the Bamboo leaves extract.    Reagent blank. For ELISA we used 50 µg protein using monoclonal antibody against human/mouse TIMP-2 (SantaCruz, USA), followed by secondary antibody coupled with HRP. TMB as substrate was used to develop the colour and reading was taken at 450 nm. For ELISA we used 50 µg protein using monoclonal antibody against human/mouse MT1-MMP (SantaCruz, USA), followed by secondary antibody coupled with HRP. TMB as substrate was used to develop the colour and reading was taken at 450 nm.

Discussion
Guava is reported to have bio active compounds to manage several diseases [1] [2]. Its anticancer effect was also reported [1] [2]. One of the major factors in cancer development is MMP-2 activity. MMP-2 activity is intimately related to the cancer development and angiogenesis. This has been studied and referred in several references [4] [12]. In our study, PBS, pH 7.4 extract of Guava Leaves show appreciable inhibition of active MMP-2 like molecule both from plant origin as well as human active MMP-2 (Breast Cancer patients' saliva). Our studies also through some light on the inhibition of activated MMP-2 like molecule derived from plant origin and also human MMP-2 (MMP-2 from human female breast cancer patient's saliva). ELISA results perhaps confirm that MT1-MMP like molecule is present in Guava leaves PBS extract. This could be a novel observation as well as could explain better why the Guava leaves extract shows anticancer effect [2]. The detail study could focus on the management of cancer.
The isolation and identification of the new MMP-2 inhibitor (other than TIMP-2) for its molecular characterization is under investigation.

Conclusion
Matrixmetalloproteinase-2 or MMP-2 is an enzyme protein very much associated with the development of cancer. It has been noticed that some inhibitor like molecule(s) may be present in the PBS extract of Guava Leaves those may inhibit the active MMP-2 like molecule of Plant origin as well as human active MMP-2. The active MMP-2 inhibitor(s) if identified may function in the management of Cancer.