A ‘Single Point’ Experimental Approach of Assessing the Inactivation Kinetics of β-glucuronidase by Aqueous-based Leaf Extractives Notably Used as Enemas

Excess beta-glucuronidase in the caecum and colon is responsible for enterohepatic circulation of toxic aglycones, which have been implicated in development of cancer, diarrhea, jaundice and other disease conditions of the gut. Botanicals are potential sources of molecules, which can serve as dietary components for inactivating beta-glucuronidase. Enzyme inactivation is employed in this study to categorize the potencies of the leaf extracts of Alstonia boonie (Ext1), Vernonia amygdalina (Ext2), Heliotropium indicum (Ext3) and Momordica charantia (Ext4) to inactivate betaglucuronidase. In a ‘single point’ experimental approach a fixed amount of five micrograms of each samples were tested for their ability to reduce β-glucuronidase activity. Extracts reduced activity of the control (100%) to about 5-30% in 20 minutes. Ext2 and Ext3 were more potent as inactivators recording lower Km, Vmax and kcat compared to Ext1 and Ext4. Ext2 and Ext3 treatments resulted in 619 and 843 fold decreases in β-glucuronidase activity respectively. The partitioning ratios kinact/Km, Original Research Article Sarfo and Abgale; JAMPS, 6(1): 1-10, 2016; Article no.JAMPS.22249 2 kcat/kinact and Di values confirmed Ext2 and Ext3 as the more potent inactivators of β-glucuronidase than Ext1 and Ext4. Furthermore, Ext2/Ext3 inactivation mechanisms may involve the formation of a tenary enzyme-inactivator-substrate complex, which could be different from the inactivation mechanisms of Ext1/Ext4. However Ext2, Ext3 and Ext4 treatment produced same magnitude of order of the inactivation reaction, n = 0.17 and inactivation rate constant, kinact = 0.0024 min -1 compared with n = 0.09 and kinact = 0.0020 min -1 of Ext1 treatment which are indication of the complexity and form of the molecules in the extracts. Thus, extracts of similar chemical entities could display varying degrees of inactivation mechanism. These results show that the ‘single point’ experimental approach can be used to categorize the potencies of consumable botanicals for their ability to inactivate β-glucuronidase.


INTRODUCTION
β-glucuronidase excreted into the intestinal lumen by the intestinal microbiota play a major role in enterohepatic circulation releasing both beneficial and toxic aglycones [1,2]. Toxic endogenous and exogenous compounds are glucuronidated in the liver and transported partly through the biliary system into the intestinal lumen to be excreted with feces [3]. Naturally, glucuronidated compounds in the intestinal lumen are poorly reabsorbed into the blood stream and efficiently eliminated from the body unless they are hydrolyzed by intestinal βglucuronidase [3]. Many of the intestinal microbiota including Ruminococus gnavus and Escherichia coli secrete β-glucuronidase into the intestinal lumen [4,5]. The greatest activity of βglucuronidase in the intestine is in the caecum and in the large intestine [5]. At these sites, the enterohepatic circulation of toxic compounds due to the activity of intestinal β-glucuronidase activity has been suggested to promote tumor formation and diarrhea in cancer patients under chemotherapy [6,7]. The enzyme has also been implicated in neonatal jaundice, the most common cause for hospital re-admission of neonates, as a result of recirculation of bilirubin after the hydrolysis of its glucuronide in the intestinal lumen [8]. Nitro-reductase and azo reductase activities in the gut also contribute to the release of aglycones, which can induce colon carcinogenesis [9]. To reduce the toxic effects of aglycones, identification of any betaglucuronidase inhibitor that can be acknowledged to be a safe dietary component could be of considerable appeal. Consequently, existing foods, drinks and beverage are being investigated for molecules that can inactivate gut enzymes whose activity can lead to inflammatory and malignant diseases. Certain components of milk and milk-based products such as yoghurt have been suggested to inhibit β-glucuronidase activity in the intestine and therefore can be used to fortify milk products to reduce enterohepatc circulation of bilirubin in neonates [9]. Multicomponent herbal drug, food and beverage preparations also hold great promise in this area of research as the one-shot treatment targeting multi-enzymes whose activity can cause cancer and inflammation of the intestinal mucosa cells. In this study a single inhibitor concentration 'single-point' inactivation kinetic parameters has been used to evaluate extracts from leaves commonly used as enemas for the presence of small molecule inactivators which may have utility as novel therapeutics against the debilitating effects of β-glucuronidase activity in the gut and to increase the dose-intensification and efficacy especially in a significant fraction of cancer patients undergoing chemotherapy.

Spectrophotometric Determination of β β β β-glucuronidase Activity
The reactions were initiated by addition of enzyme (0.01 µmoles) to a solution of 4methylumbeiliferyl-β-D-glucuronide substrate in a buffer, which has been thermally equilibrated at 27°C. β-glucuronidase was generally assayed by spectrophotometric scanning of the hydrolysis of 4-methylumbeiliferyl-β-D-glucuronide on a Jenway spectrophotometer. For the 'single point' inactivation assays, the equivalent of 5µg driedweight of each aqueous enema was used in each assay. Assays were always performed in duplicate determinations in total assay volume of 1 mL and the average absorbance values were recorded.

β β β β-glucuronidase Time-course Assay
4-methylumbeiliferyl-β-D-glucuronide (GMU) and β-glucuronidase (GUASE) stock solutions were prepared in Na 3 PO4.12H 2 O buffer. The total volume of the reaction mixture was 1mL containing a final concentration of 50 mM Na 3 PO4.12H 2 O buffer (pH 7.0), 1 mM 4methylumbeiliferyl-β-D-glucuronide and 0.01 µmoles of GUASE. The reaction was monitored for 40 minutes. Absorbance change was recorded for every minute at a maximum absorption wavelength of 350 nm. One unit of activity of GUASE was taken as the micromoles of the aglycone 4-methylumbeiliferone (MU) released into the reaction mixture per minute [10].

Michaelis-Menten Kinetic Assay
The conversion of GMU to MU by GUASE was assayed at final GMU concentration of 0.02, 0.06, 0.08, 0.16, 0.24, 0.32, 0.48 and 0.64 mM respectively. Absorbance change was recorded for every minute in a 20 minute reaction time at pH 7.0 and temperature of 27°C. Activity in micromoles per minute was calculated for each substrate concentration and the values were used to obtain the Michaelis-Menten plot of activity versus substrate concentration. K m and V max were determined from the Lineweaver-Burk plot. Duplicate measurements were taken for each preparation.

GUASE Inactivation Assay
GUASE activity was repeated for each substrate concentration (0.02, 0.06, 0.08, 0.16, 0.24, 0.32, 0.48 and 0.64 mM) after the enzyme has been incubated at 0°C in 5-microgram equivalence of each of the aqueous extract for 20 minutes respectively. Duplicate determinations were performed and the average activity was recorded for each substrate. The corresponding K m and V max values for each extract were calculated with the aid of a Lineweaver-Burk plot. Similar inactivation assay was also conducted at a fixed GMU concentration of 1 mM for each extract. Same experiments were repeated as duplicates.

Mode of GUASE Inactivation by Extracts
The 'single point' 5-µg weight equivalent of each extract was assessed for the mode of inactivation. This amount was diluted for each extract to give various preparations of increasing solubility factor (D) of 0.0, 0.1, 0.15, 0.2, 0.25, 0.35 and 0.4 in a final volume of 25 µL respectively. Solubility factor 0.4 is equivalent to the highest concentration of extract and 0.0 contains no extract. GUASE was incubated for 20 minutes at 0°C in each preparation before activity was determined by monitoring the reaction in 1 mM GMU solution for 20 minutes at 27°C. GUASE activity was recorded for each solubility factor and plotted for each extract. Duplicate measurements were taken for each preparation.

UV-spectrophotometric Scanning for the Conversion of GMU to MU
β-glucuronidase plays an important role in drug metabolism and has been used as target in a number of drug discovery studies. The enzyme cleaved 4-methylumbeiliferyl-β-D-glucuronide GMU, which has maximum absorption at 340 nm to give 4-methyumbeiliferone product MU, which absorbs maximally at 350 nm as shown in Fig. 1.

Time-course of the Reaction of GUASE
The distinct absorption peaks of the substrate GMU, and the product MU, facilitated the kinetics and inactivation studies of GUASE. In Fig. 2, 1 mM concentration of GMU was used to determine the time course for the conversion of GMU to MU.

Stability of GUASE Activity at Different Substrate Concentrations
At GMU maximal concentration of 1 mM, approximately 20 minutes was required for the conversion to MU. This time period was used to assess the kinetics of GUASE at GMU concentrations below 1 mM as shown in Fig. 3.

Kinetic Model of GUASE Activity
The Michaelis-Menten plot (Fig. 4a) of the activity of the enzyme at different GMU concentrations was also conducted at the same time period of 20 minutes.
The Lineweaver-Burk plot shown in Fig. 4b was use to estimate the parameters K m , V of the uninhibited GUASE.

Kinetic Model of GUASE Activity
Menten plot (Fig. 4a) of the activity of the enzyme at different GMU concentrations was also conducted at the same time period of plot shown in Fig. 4b was , V max and k cat

The 'Single Point' Inactivation Experiments
A time-course assay was performed after 20 minutes of incubation with extracts. This was aimed at confirming the marked reduction in activity after incubation with five micrograms equivalent weight of extract. As shown in course assay was performed after 20 minutes of incubation with extracts. This was aimed at confirming the marked reduction in activity after incubation with five micrograms equivalent weight of extract. As shown in Fig. 5, nactivation effect on the enzyme, reducing activity of the control (100%) to about 5-30% in 20 minutes.
The kinetic assay was repeated for each GMU concentration in the presence of 5 µg of each of the enemas and the reduction in activity was used to determine the change in K m , V max and k cat for each extract Extn, as shown in Table 1. The constant k inact was used to calculate the partition ratios k inact /K m and k cat /k inact as shown in Table 2.

DISCUSSION
Multi-component herbal preparations are gaining increasing prominence in medicine equally as the pure isolated mono-compounds or their synthetic drug analogs. However, the one major problem of their use has been with the difficulty of understanding the mode of action due to the multiplicity of molecules any single botanical preparation being it enzyme inhibitor/activator or receptor agonist/antagonist can present.  ability to inactivate β-glucuronidase in in vitro assay. The batch of enzyme obtained for this study showed a typical β-glucuronidase activity as shown in Fig. 1, giving distinct GMU and MU peaks at 10 nm apart. A time-dependent experiment of this activity using 1 mM GMU and 0.01 µM of β-glucuronidase produced a hyperbolic curve of Fig. 2 asymptomatic after 20 minutes of reaction. At this concentration of enzyme and fixed reaction time of 20 minutes the stability of β-glucuronidase in increasing substrate concentration was established in Fig. 3. Within this concentration range the enzyme exhibited a typical Michaelis-Menten steady state kinetic shown in Fig. 4a. The data from this kinetic were fitted to the Lineweaver-Burk equation to produce Fig. 4b, which was used to extract the Michaelis constant K m of 0.22±0.02 mM, and maximum rate of reaction V max of 0.134±0.007 µmole/min. The 'single point' involved pre-incubation (20 minutes) of βglucuronidase with a single dried mass of 5 µg obtained after evaporation of 1 mL of each of the four aqueous extracts; at fluid consistency required for use as enemas, as the inhibitory substance in each inactivation assay. In a reaction mixture of 1 mL the 5 µg of each enema material in Fig. 5 gave over 60% reduction of GUASE activity after 20 minutes of reaction. In addition, the 'single point' produced different modes of inactivation by the marked variations in the K m , V max, k cat and k cat /K m values as shown in Table 1. Ext3 (Heliotropium indicum) and Ext2 (Vernonia amygdalina) gave the lowest K m , V max and k cat . The two extracts also gave the highest fold decrease of enzyme activity compared to the control. This result is supported by the curves of Fig. 1 where the inactivation curves by Ext1 (Alstonia boonie) and Ext4 (Momordica charantia) lie above that of Ext2 and Ext3. Ext2 and Ext3 appeared to form a tenary enzymeinactivator-substrate complex during the inactivation process [13,14]. Furthermore, the catalytic efficiency of 0.533 µmole/min under Ext3 treatment was an improvement over the other extracts, however Etx3 in the same reaction gave the highest value of fold decrease of 843 compared to 409 of Ext4 treatment, which corresponds to the lowest catalytic efficiency of 0.015 µmole/min. These events are indicative of the different modes of inactivation by the extracts. The extents of these mechanisms by the 'single point' approach are depicted by the marked variations in the partition ratios k inact /K m and k cat /k inact [14], in Table 2. Ext2 and Ext3 gave the lowest k cat /k inact values and highest k inact /K m values, which further support the two extracts as stronger inactivators of β-glucuronidase than Ext1 and Ext4. The partitioning ratios also show some levels of relatedness between Ext1 and Ext4 and between Ext2 and Ext3 on mechanism of enzyme inactivation. This study introduced a solubility parameter, which reflects the necessity to obtain   enemas. The use of Di is analogous to K i ; lower Di value corresponds to an effective enema that can inactivate GUASE in a very diluted state and the vice versa. Di of Ext2 and Ext3 were lower than Di values of Ext1 and Ext4; an additional proof of the effectiveness of the two extracts to inactivate β-glucuronidase. In spite of the above relationships, the order of reaction n and rate of inactivation k inact parameters present Ext1 in different category of inactivators compared to Ext2, Ext3 and Ext4 as shown in Table 2. Values of k inact were similar for all extracts which confirm the rapidity of the inactivation process observed in Fig. 5. This justified the use of the partioning ratios k inact /K m , k cat /k inact and the solubility constant Di as the true reliable parameters to compare the inactivation rates of the extracts [15]. The order of reaction n, however placed Ext1 in a different category compare to Ext2, Ext3 and Ext4; a strong indication that Ext2, Ext3 and Ext4 might present complexity of metabolites, which is different from Ext1 but the pairs Ext1/Ext4 and Ext2/Ext3 share the same mode of βglucuronidase inactivation respectively.

CONCLUSION
A herbal medicine with a complex set of compounds presents a unique difficulty in understanding its mechanism of action. This understanding may be even more daunting when such mechanism is studied for a multifactorial disease; disease condition where a multicompound-medicine may have advantage over a single-compound medicine in a multi-target therapy approach. Complex diseases may sometimes be triggered by one-step pathway. In such situation inactivating the enzyme/receptor involved in this pathway will prevent the occurrence of a disease and the multiplicity of bioactive and non-active molecules in a medicine may not be an issue in the understanding of the mechanism of such inactivation process. Inactivation of β-glucuronidase in the gut to avoid enterohepatic circulation of toxic aglycones may represent the latter scenario. In this study partitioning ratios k inact /K m , k cat /k inact , fold decrease of enzyme activity, solubility constant, Di, and the order of the inactivation reaction, n have been used to categorize four different enemas of their potential to inactivate betaglucuronidase activity. The 'single point' approach did not require the prior knowledge of the form or concentration of the active and nonactive components. It was possible by this approach to evaluate the inactivation without having to depend on any active molecule and therefore avoid the usual ranking parameters K i and IC 50 , which depend on the reversibility of the inhibition/inactivation and a saturation inhibition/inactivation kinetic. Ext3 is potent inactivator, which is possible to involve an irreversible formation of a tenary enzymeinhibitor-substrate complex. Ext2 may have a lesser degree of this mechanism. Ext1 and Ext4 have inactivation mechanism different from Ext2 and Ext3, even though Ext2, Ext3 and Ext4 appeared to have the same complexity of metabolites, which may be different from Ext1. Consumable botanicals; foods and medicines can be screened with this approach to categorize them in the search for food and micronutrient supplements to serve as blocking agents in the special conditions of jaundice in neonates, patients with chemotherapy induced-diahrroea, cancer development in the colon and other similar conditions, which are triggered by enterohepatic circulation.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.