Biotransformation of Gallotannins From Fresh Fruit Juice of Emblica officinalis
in In-vitro System
Snehal S. Patel
Ramesh K. Goyal
Gallotannins, esters of carbohydrates or polyols with gallic acid present in
fresh fruit juice of Emblica officinalis and has been found to be pharmacologically
active. The knowledge of dietary intake of gallotannins and their bioaccessibility
in gastrointestinal track are key factors in assessing their significance in
human health. This study illustrates a biotransformation of gallotannins from
fruit juice in gastrointestinal fluids and analysis of bioactive marker by rhodanine
assay. Total gallic acid was determined by rhodanine assay from fruit juice
of Emblica officinalis. Gastric juice and intestinal fluid caused hydrolysis
of fruit juice during incubation for 8 h. The hydrolysis product from fruit
juice was gallic acid. In vitro studies indicated that dissociation of
gallic acid from ester complexes was apparent in both the fluid but gallotannins
were rapidly hydrolyzed in gastric juice rather than intestinal fluid. Thus,
our data suggest that hydrolysis of gallotannin in gastrointestinal fluid produced
gallic acid which is possibly responsible for pharmacological effects.
to cite this article:
Snehal S. Patel and Ramesh K. Goyal, 2013. Biotransformation of Gallotannins From Fresh Fruit Juice of Emblica officinalis
in In-vitro System. Research Journal of Phytochemistry, 7: 18-23.
Received: June 27, 2012;
Accepted: March 09, 2013;
Published: June 29, 2013
Gallotannins are polyphenolic compounds found in legumes, vegetables, fruits
and beverages and are the most abundant antioxidants in our diets. Gallotannins
were reported to possess multiple biological activities including anticancer,
antioxidant, antiallergic, anti-inflammatory, antihyperglycaemic, lipid lowering
and antimicrobial activities and their consumption may contribute to prevent
stroke, cardiovascular heart disease, neurodegenerative diseases (Manach
et al., 2005; Bhattacharya et al., 2002;
Mathur et al., 1996; Patel
and Goyal, 2011a).
Emblica officinalis Geart. (Family: Euphorbiaceae) commonly known as
Amla or the Indian gooseberry, have been reported to contain constituents
with variable biological activity. Phytochemical investigations of fruits of
Emblica officinalis show that it is having high amount of polyphenol
content like low and high molecular weight gallotannins such as L-malic acid
2-O-gallate, mucic acid 2-O-gallate, mucic acid, 1-O-galloyl-β-D-glucose,
mucic acid 6-methyl ester 2-O-gallate, mucic acid 1,4- lactone 2-O-gallate,
mucic acid 1-methyl ester 2-O-gallate, mucic acid 2-O-gallate, mucic acid 1,4-lactone
6-methyl ester 2-O-gallate, mucic acid 1,4-lactone 3-O-gallate, mucic acid 1,4-lactone
3,5-di-O-gallate, corilagin (Fig. 1) (Zhang
et al., 2001; Kumaran and Karunakaran, 2006;
Anila and Vijayalakshmi, 2002; Ghosal
et al., 1996). Gallotannins present in number of medicinal plants
reported to possess various pharmacological activities (Patel
and Goyal, 2012; Rahman et al., 2009).
|| Structures of gallotannins from fruit juice of E. officinalis
The gallotannins are hydrolysable tannins which contain a central core of
carbohydrates which are esterified by phenolic like gallic acid and, unlike
condensed tannins are readily hydrolyzed by acids, bases, or certain enzymes
(Salunkhe et al., 1989: White,
1957). On this basis, gallotannins is expected to be subject to degradation
within the gut of mammals. Gallotannin which has esterified to gallic acid,
is expected that, like other hydrolysable tannins, gallotannin will release
those gallic acid residues in free form upon hydrolysis. On this basis, possible
hydrolysis of gallotannin, resulting in the release of gallic acid, during incubation
with gastrointestinal fluid, the present investigations was carried out in the
simulated gastric and intestinal fluid and release of gallic acid residue in
free form upon hydrolysis was determined by rhodanine assay. Despite many biological
activities of gallotannins no data available for bioavailability of gallotannins
from fruit juice in gastrointestinal tract. Thus aim of this present work was
to estimate the amount of total gallotannin consumed in a fruit juice of Emblica
officinalis and their gastrointestinal bioaccessibility.
MATERIALS AND METHODS
The fresh fruits of Emblica officinalis Gaertn. were purchased in the
month of January of 2008 from Gaziabad, Madhypradesh, India and authenticated
by Department of Pharmacognosy, L.M.College of Pharmacy, Gujarat University,
Ahmedabad, India and study was carried out for period of one week.. Juice from
fruits was freshly prepared in our laboratory before study. Other chemicals
used were of analytical reagent grade.
Preparation of simulated gastric juice: Sodium chloride (2.0 g) and purified pepsin (3.2 g), with an activity of 8000 to 2500 units per mg of protein dissolved in 70 mL of 1 M HCl. Sufficient water was added to make 1000 mL and the pH of resulting solution was adjusted to 1.5 with HCl.
Preparation of simulated intestinal juice: Monobasic potassium phosphate was dissolved in 250 mL of water. The solution was mixed with 77 mL of 0.2 N NaOH and 500 mL of water. Pencreatin (10 g) was mixed to the solution. The pH of resulting solution was adjusted 6.8 either with 0.2 N NaOH or 0.2 N HCl and diluted with sufficient water to make 1000 mL.
Incubation of fruit juice of E. officinalis with simulated gastric juice and intestinal juice: Equal volumes (10 mL) of simulated gastric or intestinal juice and fruit juice of E. officinalis were mixed; the mixtures were incubated at 37°C under anaerobic condition. During incubation, 1 mL of sample was withdrawn at different time interval up to 8 h. The sample was used for gallic acid determination as described below.
Determination of gallic acid by rhodanine assay: The hydrolysate was
diluted to appropriate concentration for determination of gallic acid. 200 μL
of blank, hydrolysate and standard were mixed with 0.3 mL 0.667% methanolic
rhodanine solution. After exactly 5 min, 0.2 mL of 0.5 N aqueous KOH solution
was added. After 2.5 min. the mixture was diluted to 5.0 mL with distilled water.
Five to 10 min later the absorbance was read at 520 nm. The total gallic acid
content was expressed as milligrams of gallic acid/1 mL fruit juice (Inoue
and Hagerman, 1988).
The biological properties and bioavailability of gallotannins depend on their chemical structure and it is important to study the effect of various pH conditions in gastrointestinal tract. As shown in Table 1, gallotannins present in fruit juice of E. officinalis hydrolyzed and generate free gallic acid in simulated gastric juice (3.5 mg mL-1) and intestinal fluid (2.1 mg mL-1). The hydrolysis of gallotannins by simulated gastric juice was relatively rapid and stable over the course of 8 h. On the other hand, hydrolysis of gallotannins by intestinal fluids was slow as compared to gastric juice. The amount of gallic acid produced reached a plateau after 1.5 h of incubation with simulated gastric juice at 37°C. However, plateau was reached after 4 h of incubation with simulated intestinal fluid. Therefore, it is presumed that the administered gallotannins present in fruit juice of E. officinalis hydrolyzed to gallic acid by simulated gastric juice and there after in intestinal fluid by acting on the gallic acid esters of gallotannins. Total contents of gallic acid in the fruit juice were found to be 24.3 mg mL-1. Fruit juice contained no gallic acid before incubation either in gastric juice or intestinal fluid.
|| Amounts of gallic acid released over 24 h after incubation
of fruit juice of E. officinalis
|nd: Not detected
Tannins are phenolic compounds that occur widely in the plant kingdom. They
are broadly classified into two groups based on chemical structure: hydrolyzable
tannins and condensed tannins. Hydrolyzable tannins contain a central core of
carbohydrates (glucose and polyhydric alcohol). which are esterified by phenolics
(gallic acid, ellagic acid) and, unlike condensed tannins, are readily hydrolyzed
by acids, bases, or certain enzymes. Figure 1 shows gallotannins
present in fruit juice of Emblica officinalis such as L-malic acid 2-O-gallate,
mucic acid 2-O-gallate, corilagin chebulagic acid, putrajivain A, elacocarpusin,
mucic acid,1-O-galloyl-β-D-glucose, mucic acid 6-methyl ester 2-O-gallate,
mucic acid 1,4- lactone 2-O-gallate, mucic acid 1-methyl ester 2-O-gallate,
mucic acid 2-O-gallate, mucic acid 1,4-lactone 6-methyl ester 2-O- gallate,
mucic acid 1,4-lactone 3-O-gallate, mucic acid 1,4-lactone 3,5-di-O-gallate,
emblicanin A and B, punigluconin, pedunculagin, methyl gallate, corilagin, furosin
and geraniin. It has been demonstrated by several workers that pH and action
of digestive enzymes are the most important factors affecting the hydrolysis
of hydrolyzable tannin like gallotannins (Van Buren and
Robinson, 1969; Berenbaum, 1980; Martin
et al., 1985; Saura-Calixto et al., 2007).
Gallotannins are found in legumes, vegetables, fruits and beverages and reported
to possess multiple biological activities (Mishra et
al., 2011). Phytochemical investigations of fruits of E. officinalis
shows that it is having high amount of polyphenol content like low and high
molecular weight gallotannins. Therefore to elucidate the significance of gallotannins
in human health, it is essential to know the amount of gallotannins in the fruit
juice of E. officinalis and their bioavailability.
The effects of consumption of fruit juice of E. officinalis on health
have recently received a great deal of attention. Our laboratory studies have
demonstrated antidiabetic and cardioprotective activity of fruit juice in different
animal models (Patel and Goyal, 2011a, b).
Phytochemical investigation revealed that fruit juice possessed above pharmacological
activity may be due to higher concentration of polyphenols like gallotannins.
The molecular mechanisms for these actions are under investigation. A major
problem in investigating the relationship between fruit juice and antidiabetic
effect is the lack of quantitative data. Even in studies with animals, mechanistic
understanding of the effect of fruit juice against antidiabetic activity is
hampered by a lack of information on the bioavailability of the effective components
of it. It was found that the gallotanins present in E. officinalis is
essential for their potent antioxidative and antidiabetic activities. Gallotannins
are hydrolysable tannins which may get hydrolyzed into free form of gallic acid
in gastrointestinal tract. Gallic acid is the biological marker compound present
in the form of esters in fruit juice of E. officinalis (Patel
and Goyal, 2011b). It is reasonable to speculate gallotannin present in
E. officinalis are hydrolyzed to gallic acid which in turn produces various
pharmacological activities. In the present study, fruit juice of E. officinalis
incubated with gastric and intestinal fluids of gastrointestinal tract and caused
generation of free gallic acid in simulated gastric juice and intestinal fluid
by dissociation of the bond between gallic acid and carbohydrate (Fig.
2a). However, hydrolysis with gastric juice was much faster than intestinal
fluid after 8 h of incubation (Fig. 2b). It was shown that
after 24 h of incubation concentration of gallic acid found to be 16.6 and 8.5
mg mL-1 in gastric and intestinal fluids respectively (Fig.
2a, b). Our result of the study are in coordinance with
previous reports where in vivo metabolism of esters of gallic acid like propyl
and lauryl gallate was carried out and major metabolite obtained was gallic
acid (Booth et al., 1959). The metabolic behavior
of orally administered esters of gallic acid was investigated by several scientists
on various animals like rat, rabbit chicken as well as on human being (Zong
et al., 1999; Watanabe and Oshima, 1965;
Potter and Fuller, 1968; Shahrzad
et al., 2001; Shahrzad and Bitsch, 1998).
||Concentration of gallic acid in simulated gastric juice and
intestinal fluid at different time intervals
They have examined effect of change in pH resembling those of stomach by measuring
the amount of gallic acid released from tannic acid. The results of our study
were consistent with their findings. Thus, it can be postulated that, gastric
juice facilitated the hydrolysis of gallotannin, as judged by the release of
its constituent gallic acid. Thus, it can be postulated that, if fruit juice
of E. officinalis were consumed, gallic acid would be readily liberated
from the ester complexes in the acidic stomach or the alkaline small intestine
and thus could be available as sources of nutrients.
This study was supported by National Facilities in Engineering and Technology with Industrial Collaboration (NAFETIC) scheme sponsored by AICTE, New Delhi, India.
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