Ancient Science of Life

: 2015  |  Volume : 34  |  Issue : 3  |  Page : 134--141

Evaluation of effects of Maṇḍurabhasma on structural and functional integrity of small intestine in comparison with ferrous sulfate using an experimental model of iron deficiency anemia

Suchita Rajanikant Gawde, Tejal C Patel, Nirmala N Rege, Snehalata Gajbhiye, Dinesh Uchil 
 Department of Pharmacology, Seth G.S. Medical College, KEM Hospital, Parel, Mumbai, Maharashtra, India

Correspondence Address:
Dr. Suchita Rajanikant Gawde
1/64, Anurag, Datar Colony, Bhandup East, Mumbai - 400 042, Maharashtra


Background: The present study was planned to assess effects of Maṇḍurabhasma (MB) on structural and functional integrity of small intestine using an animal model of iron deficiency anemia (IDA) in rat. Methods: IDA was induced by giving iron deficient diet and retro-orbital bloodletting for 21 days in Wistar female rats. Rats (n = 72) were divided into six groups: (i) Control group, (ii) IDA rats, (iii) IDA rats receiving vehicle, (iv) rats receiving ferrous sulfate (40 mg/kg), (vi) rats receiving a low dose (22.5 mg/kg) of MB, (vi) rats receiving a high dose (45 mg/kg) of MB. Treatment was conducted for a period of 21 days followed by an assessment of change in hemoglobin (Hb) levels, lactase levels, lipid peroxidation activity by measuring malondialdehyde (MDA) levels and jejunal morphometry. Results: In the present study, the lactase activity was markedly reduced in iron-deficient rats. Our study has demonstrated that intestinal morphology and MDA levels were not altered in the animals with IDA as compared to normal animals. In phase II, improvement in Hb response to ferrous sulfate was accompanied by an improvement in lactase activity. However, it significantly increased MDA levels with derangement of the normal villous structure. Rats receiving a low dose of MB did not have increased MDA levels. It did not alter the jejunal villous structure and improved lactase activity, but hematinic activity was found to be less than that of ferrous sulfate. Rats receiving a high dose of MB showed significantly improved Hb as well as lactase levels. They exhibited damage to the villous structure and increased MDA levels, but the effects were significantly less as compared to ferrous sulfate group. Conclusion: Rats receiving a high dose of MB have shown improvement in hematinic and lactase levels comparable to those receiving ferrous sulfate. However, it causes lesser oxidative damage as compared to ferrous sulfate. This is an encouraging finding because it indicates the potential of MB to cause lesser gastrointestinal side effects compared to ferrous sulfate.

How to cite this article:
Gawde SR, Patel TC, Rege NN, Gajbhiye S, Uchil D. Evaluation of effects of Maṇḍurabhasma on structural and functional integrity of small intestine in comparison with ferrous sulfate using an experimental model of iron deficiency anemia.Ancient Sci Life 2015;34:134-141

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Gawde SR, Patel TC, Rege NN, Gajbhiye S, Uchil D. Evaluation of effects of Maṇḍurabhasma on structural and functional integrity of small intestine in comparison with ferrous sulfate using an experimental model of iron deficiency anemia. Ancient Sci Life [serial online] 2015 [cited 2022 Jul 1 ];34:134-141
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Iron deficiency anemia (IDA) is a major public health problem in India. In the gastrointestinal (GI) tract, IDA can cause stomatitis and glossitis, [1],[2] reduction of gastric acid secretion, [3] intestinal atrophy and abnormal absorption. [4] It has been shown on many occasions that dietary iron deficiency can cause a significant decrease in the activity of enzyme disaccharidase especially lactase [5],[6],[7] and that this reduction in enzyme activity is known to be reversed by iron supplementation. [8],[9],[10]

Management of IDA requires correcting the cause first, wherever possible, along with replacing body iron stores which is achieved by administering ferrous salts. Although ferrous salts rapidly correct the iron deficiency, they cause free-radical-mediated mucosal damage. [11] Excess iron remains in the body and participates in Fenton reaction leading to the production of reactive oxygen species (ROS) causing cellular damage. [12],[13] Mucosal injury is induced by the initiation of lipid peroxidation either by iron itself or by the ROS produced during the Fenton reaction. Lipid peroxidation of cell membranes, including mitochondrial membranes, can in turn compromise cellular integrity and function and affect its energy status, thereby causing further tissue injury. [14]

Oral iron therapy has several GI side effects, such as nausea, vomiting, hyperacidity, abdominal pain, and constipation. [15] Iron-induced oxidative damage in the intestine after oral ingestion of iron supplements may, in part, be responsible for these GI side effects. [16] Hence, the treatment of IDA needs a hematinic which will not only correct the iron deficiency but also cause less oxidative damage resulting in better tolerability.

The Ayurvedic iron preparation - Maṇḍurabhasma (MB) has been used since ages in India for treating anaemia (pāṇḍuroga). [17] It is also recommended in Ayurveda for the treatment of menorrhagia, amenorrhea, dysmenorrhea, chlorosis, diarrhea, dyspepsia, kidney diseases, albuminuria, and nervous diseases. [18] It has also been shown to be valuable in the treatment of hemolytic jaundice and microcytic anemia. [19]

A previous study conducted in our department has confirmed that MB exhibits hematinic activity comparable to that of ferrous fumarate in a rat model of IDA. [20] However, the effect of MB on structural and functional integrity of intestine remains to be evaluated. Hence, the present study was conceived to carry out a comparative evaluation of MB and ferrous sulfate on variables like lactase levels, jejunal morphometry, and lipid peroxidation activity in IDA.


The entire study was divided into two phases. Phase I involved evaluation of the effect of IDA on intestinal lactase activity, lipid peroxidation, and jejunal morphometry. Phase II involved comparing the effect of Ayurvedic haematinic preparation - MB on intestinal lactase, lipid peroxidation, jejunal morphometry with that of ferrous sulfate.

Ethical considerations

The study was undertaken after obtaining the permission of the Institutional Animal Ethics Committee (AEC/01/2011). The guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) were followed during the entire study.

Experimental animals

Rats of Wistar strain (female, n = 72) weighing 120-180 g and of 6-8 weeks age were selected for the experiment.

Husbandry conditions

Rats were procured from the in-house colony available in the Centre for Animal Studies of the Institution. They were maintained throughout the study period in accordance with the guidelines for the Care and Use of Laboratory Animals laid down by the CPCSEA, India. These rooms had 12-15 filtered fresh air changes per hour, 22 ± 3°C temperature and 30-70% relative humidity. Twelve hourly light and dark cycles were maintained. Animals were housed in groups of four animals per cage in polypropylene cages. Autoclaved paddy husk served as the bedding. Cages were fitted with stainless steel top grill having facilities for attachment of feeding bottles. Study groups received milk and no other diet for the whole duration of study except animals in normal control group which were given commercially available rodent food (VRK Nutritional Solutions, Pune) and filtered and ultraviolet purified water ad libitum.


In phase I, 24 rats were randomly divided into two groups, each comprising 12 animals. The hemoglobin (Hb) levels were assessed on Day 0. One group served as the normal control group and was allowed normal rat diet and water ad libitum (Group N). The rats in the other group were bled (1 ml) daily through puncture of the retro-orbital sinus for the period of 21 days (Group D). In addition, Group D received milk diet (400 ml/cage/day/4 rats) instead of a normal diet. On day 21, estimation of Hb levels was done in both the groups to confirm IDA in Group D. The rats from Group N and Group D were further sub-divided into two groups of six animals each. In sub-group 1, intestinal mucosal lactase levels were assessed by the method described by Dahlqvist [21],[22] and in the sub-group 2, jejunal mucosa was processed for estimation of a biomarker of lipid peroxidation and for morphometry. Lipid peroxidation activity was assessed by measuring malondialdehyde (MDA) levels by the method described by Ohkawa et al. [23] Under light microscopy, all the villi observed in the sections were classified according to Lee and Toner's classification [24] into finger-like villi, leaf-like villi, blunted/ridged villi. The percentage of villi belonging to each class was calculated. Morphometric examination was carried out using a trinocular microscope with an image analyzer Image-Pro ® Plus Version 6.2 for Windows™ (Media Cybernetics, MA, USA) which permitted measurements of following variables: Villous height, total mucosal thickness, crypt depth, villous/crypt ratio, enterocyte height for each rat. Measurements were taken from 10 different sites of the histopathological slide. The height of 20 enterocytes was measured in the middle third of the villous.

In phase II, rats (n = 48) were bled 1 ml of blood (retro-orbital route) daily for 21 days and provided milk as a sole dietary item. After confirming IDA on day 21, they were randomly allocated to four treatment groups (n = 12 rats/group). Group 1 served as the vehicle control (carboxymethyl cellulose [CMC] 0.5%); Group 2 received standard therapy in form of ferrous sulfate (40 mg/kg); Groups 3 and 4 were the Test groups and received MB in doses of 22.5 mg/kg and 45 mg/kg, respectively. The vehicle/drug therapy continued for a period of 21 days. During this period, the bleeding from the retro-orbital sinus was discontinued but the dietary source was only milk.

At the end of the treatment period, the groups were divided into two groups of six animals each. In the first group, lactase levels were assessed and in second group, lipid peroxidation was assessed, and jejunal morphometry was conducted as described above.

Statistical analysis

The generated data were recorded using Microsoft ® Office Excel 2007. Statistical analysis was done using the IBM SPSS software (version 16.0, IBM, Chicago, USA). The significance level was set at P < 0.05. The statistical tests used for analyzing the data are as follows:

Phase I

Unpaired t-test was used to compare effects of IDA on the following variables: Hb levels, lactase activity, MDA levels, villous morphology, and villous heights.

Phase II

A one-way analysis of variance with post-hoc Tukey's test was used to compare the effects of the drugs on the following variables: Hb levels, lactase activity, MDA levels, villous morphology, and villous heights.


In phase I, baseline Hb levels were estimated in both Groups N and D, which were comparable. At the end of induction period of 21 days, Hb levels were significantly reduced (P < 0.001) in Group D (Hb - 6.85 ± 0.32 g/dl) compared to Group N (Hb - 14.11 ± 0.68 g/dl).

Variables assessed in normal and iron deficiency anemia groups

Lactase enzyme activity of jejunum

As shown in [Figure 1], the lactase enzyme activity of jejunum observed in the iron-deficient group was found to be significantly less as compared to the normal control group (P < 0.001).{Figure 1}

Jejunal malondialdehyde levels

As shown in [Figure 2], the MDA levels of jejunum observed in IDA though showed a decrease but the difference was not statistically significant.{Figure 2}

Histopathology of jejunum

Villous morphology

Villous morphology of both the groups is summarized in [Table 1]. It was observed that both in the normal control group and IDA group, the maximum number of villi were finger-like villi, followed by leaf-like, followed by blunted/ridged villi. No significant difference was observed in the percentage of finger-like villi, leaf-like villi, and blunted/ridged villi in the jejunum in both the groups.{Table 1}

Jejunal morphometry

No significant difference was observed in villous heights, crypt heights, villi crypt ratio, mucosal thickness, and enterocyte height in both the groups [Table 2].{Table 2}

In phase II, at the end of 21 days treatment period, mean Hb levels in ferrous sulfate (14.03 ± 0.6 mg/dl) and MB high dose (14.38 ± 0.25 mg/dl) group were found to be significantly higher (P < 0.05) as compared to CMC group (8.98 ± 0.5 mg/dl) and were comparable to control group (13.86 ± 0.76 mg/dl). However, the mean Hb level in MB low dose (10.13 ± 0.76 mg/dl), remained lower than those of control and was comparable to CMC [Figure 3].{Figure 3}

Variables assessed in treatment groups

Lactase enzyme activity of jejunum

As shown in [Figure 4], the lactase enzyme activity of jejunum observed in ferrous sulfate (5.10 ± 0.88 μmol/g), MB low dose (3.78 ± 0.84 μmol/g), and MB high dose group (4.22 ± 0.6 μmol/g) was found to be significantly higher as compared to the CMC group (2.58 ± 0.33 μmol/g) (P < 0.05) and were comparable to control group (4.33 ± 1.07 μmol/g).{Figure 4}

Jejunal malondialdehyde levels

Though MDA levels in ferrous sulfate (291.46 ± 27.59 nmol/g) and MB high dose group (221.75 ± 29.06 nmol/g) were significantly higher when compared with CMC (139.12 ± 22.37 nmol/g), MDA level in MB high-dose group was significantly lower compared with ferrous sulfate. MDA levels (173.41 ± 20.76 nmol/g) in MB low dose group were comparable to CMC group.

Histopathology of jejunum

Villous morphology

Villous morphology of all treatment groups is summarized in [Table 3]. It was observed that in the CMC group, the maximum numbers of villi were finger-like, [5] followed by leaf-like, followed by blunted/ridged villi. However, in ferrous sulfate and MB high dose group, the maximum numbers of villi were the blunted/ridged villi which are significantly higher compared to CMC group (P < 0.01). The presence of finger-like villi was less in all treatment groups compared to CMC group as shown in [Table 3].{Table 3}

Jejunal morphometry

It was observed that ferrous sulfate group showed a significant reduction (P < 0.01) in all parameters, that is, villous height, villous crypt ratio, mucosal thickness and enterocyte height except crypt depth. MB low-dose group showed significant reduction of villous height and villous crypt ratio when compared to CMC group. However, reduction of villous height and villous crypt ratio was significantly lower (P < 0.05) when compared to ferrous sulfate group. Mucosal thickness, Crypt depth and enterocyte height were comparable to CMC group, and mucosal thickness was significantly higher when compared to ferrous sulfate group as shown in [Table 4].{Table 4}

Maṇḍurabhasma high dose exhibited significant reduction (P < 0.05) of villous height, mucosal thickness, and villous crypt ratio when compared to CMC group, reduction in the above-mentioned parameters was significantly less (P < 0.05) when compared to ferrous sulfate. No reduction in enterocyte height and crypt depth was observed in the MB high dose group when compared to CMC group [Table 4].


Bloodletting has been included among various models used to induce anemia in animals. [25] In the present study too, when the animals underwent daily bloodletting for 21 days along with an iron-deficient diet, Hb level dropped significantly. These levels were found to be comparable to those reported earlier. [20] This finding validates the model of IDA standardized in our department in the previous work carried out on anemic animals. Hence, two insults viz., inadequate iron intake and chronic blood loss were used together in order to induce IDA.

Malabsorption that results following IDA is mainly attributed to a significant reduction in lactase activity [6],[7],[9],[10],[26],[27],[28] and this decrease is reversible after iron supplementation. [8],[9],[10] Furthermore, ferrous salts, commonly used in the treatment of IDA may induce oxidative stress due to their catalyzing role in Fenton reaction, resulting in the production of highly reactive hydroxyl radicals. [12],[13] These free radicals generated give rise to a cascade of reactions which damage cellular lipids, proteins, and DNA. Excessive production of free radicals causes disturbance in the pro- and anti-oxidant balance, resulting in oxidative damage. [29]

We therefore, primarily assessed the effect of IDA on intestinal disaccharidase activity, lipid peroxidation by measuring jejunal MDA levels and jejunal morphometry.

In IDA, lactase enzyme activity is affected more than that of sucrase and maltase levels. [5] Hence, in our study, when we estimated the lactase enzyme activity in jejunum, it was found to be markedly reduced in anemic animals. Reduction in lactase activity in iron deficient animals, as shown by earlier workers, has been confirmed in the present study. [7],[10] This also is in agreement with other studies which also reported a reduction in the activity of jejunal disaccharidase in dogs with iron deficiency. [28]

Iron deficiency may probably be responsible for a functional alteration of enterocytes, with reduced ability to synthesize lactase. An earlier study explored the mechanism responsible for decrease in disaccharidase activity seen during iron deficiency and concluded that the reduction in enzyme levels is not caused by a reduced ability to synthesize membrane-bound glycoproteins but rather due to an alteration of the expression of the genes encoding the enzymes. [5] This reduction in gene expression was most likely caused by repression of lactase promoter regions by PDX-1. This could be the probable explanation for reduced lactase enzyme activity in iron deficient rats.

In our study, when lipid peroxidation activity (MDA levels) of the small intestine in anemic rats was compared with the normal group, no significant change was observed. Díaz-Castro et al. has reported similar findings in their study stating that lipid peroxidation activity was similar in liver cytosols from IDA and control rats indicating iron deficiency did not influence lipid peroxidation in rats. [30]

The studies which have used intestinal lipid peroxidation activity as a marker of oxidative damage are sparse. In a study conducted at NIN Hyderabad, unaltered lipid peroxidation activity in IDA has been demonstrated. [31] Our findings corroborate the same, which means iron deficiency exerts no oxidative stress on the intestinal mucosa. However, in a study conducted by Ghada et al. [32] evaluating the effect of IDA (3 weeks) followed by treatment with ferrous sulfate (40 mg/kg for 6 weeks) on lipid peroxidation activity in various tissues e.g., liver, kidney, plasma significant reduction in liver and kidney MDA and slight reduction in plasma MDA in iron-deficient rats as compared to normal rats was observed. In contrast to animal studies, human data demonstrate an increase in lipid peroxidation in anemic patients, which reduces significantly after treatment. [33],[34]

Our study demonstrates that the intestinal morphology was not altered in the animals with IDA as compared to normal animals. Similar findings have been reported by several researchers using rats as experimental animals. [6],[7],[9] They concluded that the small intestinal morphology of animals with IDA was rarely different from normal, even with severe iron depletion. Fernandes et al., justified the above finding by demonstrating the absence of differences in cell proliferation in iron deficient and normal group. [9]

It is possible that under controlled laboratory conditions, the IDA in laboratory animals do not show any structural changes in the small intestine in contrast to findings seen in clinical studies. [6],[7],[9] Wayhs et al. however showed anemic rats had a significantly greater villous height, total mucosal thickness and surface length than the controls. [35] They explained that the results probably could be attributed to the compensatory mechanisms of the intestine so as to facilitate iron absorption.

In phase II of our study, after 3 weeks of therapy the ferrous sulfate group, showed an increase in the Hb levels that reached within the normal range for rats. This rise was higher than that seen in the vehicle-treated group indicating that it is not just the stoppage of bleeding but also supplementation of iron that resulted in attaining normal values. Hematinic response of MB high dose (45 mg/kg) was comparable to that of ferrous sulfate but not low dose (22.5 mg/kg). Thus, though both the doses are advocated in Ayurveda to treat anemia in clinical practice, it is the high dose which showed a good hematinic response which was comparable to that of ferrous sulfate.

Treatment with ferrous sulfate increased lactase enzyme activity which is in agreement with previous studies. [8],[9],[10] It also significantly increased intestinal MDA levels causing oxidative damage to the intestinal mucosa. Despite the extensive literature on iron and lipid peroxidation, only two studies have investigated the effects of oral iron supplements on lipid peroxidation in the small intestine. One of them was a study conducted by Srigiridhar and Nair which showed that the intestinal MDA levels of iron treated anemic rats was found to be 2 times more than untreated anemic rats. [13] Another study conducted at NIN Hyderabad demonstrated that iron supplementation resulted in significant increase in MDA levels. [31] Ghada et al. also showed a significant increase in the liver, kidney, and plasma MDA levels after 6 weeks of treatment with ferrous sulfate (40 mg/kg). [32]

In addition, treatment with ferrous sulfate altered the jejunal villous morphology and in that it induced blunting of villi, reduced villous height and enterocyte height, decreased mucosal thickness as well as villous crypt ratio. In a previous study conducted at NIN Hyderabad, duodenum of iron-deficient rats showed a reduction in villous height and complete erosion of microvillus. [31] These effects were shown to be mediated through iron-induced hydroxyl radicals produced within the micro-environment of the GI tract. Thus, as expected, ferrous sulfate caused oxidative damage affecting intestinal mucosal barrier but at the same time it increased lactase enzyme levels.

Maṇḍurabhasma, both at high and low doses significantly increased lactase enzyme levels comparable to ferrous sulfate. Thus, MB appeared to improve the functional status of the villi for carbohydrate digestion by enhancing lactase levels.

Malondialdehyde levels in MB low dose group did not show any significant increase, which indicates less oxidative damage. On jejunal morphometry, this dose was found to cause significantly less damage to the villi and mucosal thickness. However, in this group, the Hb levels also did not show a significant rise. Thus, though this dose appeared to be safe but found to be not optimum in effectiveness.

On the other hand, high dose of MB increased MDA levels of the jejunum; but the rise was significantly lower as compared to ferrous sulfate. These findings suggest that Maṇḍurabhasma has less propensity to induce oxidative damage as compared to ferrous sulfate.

With the high dose of MB, the percentage of blunted villi as measured by the histopathological examination was found to be comparable to that of ferrous sulfate group. However, the measurement of villous height, mucosal thickness, and villous crypt ratio measured by image analyzer were found to be significantly lower as compared to the ferrous sulfate group. Similarly, no reduction in enterocyte height and crypt depth was observed in MB high dose group. These findings support that the high dose of MB was effective and yet had better safety profile than ferrous sulfate.

The point to note is that there was an improvement in lactase activity despite the structural and cellular damage caused by MB high dose and ferrous sulfate. The decrease in lactase activity seen during iron deficiency is due to an alteration of the expression of the genes encoding the enzymes; [5] hence reversal of the lactase enzyme activity shown by MB and ferrous sulfate can be attributed to the role of iron in increasing gene expression. The lactase activity depends upon genetic expression of the gene encoding the enzyme rather than the structural alteration of the villous per se Based on this, one can explain the seemingly contradictory effects on lactase levels and villi damage.

Maṇḍurabhasma in high dose appeared to be a good hematinic agent comparable to the commonly used iron preparation, that is, ferrous sulfate. In addition to this, it also showed less propensity to cause structural damage, exhibiting its protective role in preserving the structural integrity of GI tract. This is an encouraging finding because it indicates less potential of MB to cause GI side effects as compared to ferrous sulfate. It is in accordance with the claims mentioned in standard Ayurvedic texts about the use of MB - a drug with better tolerability and hence better compliance. [19]

The present study, therefore, provides a rationale for the use of 500 mg/day of MB in clinical practice which has exhibited hematinic effect comparable to that of ferrous sulfate. MB in a dose of 250 mg/day has been recommended by Ayurvedic literature to be used in the treatment of IDA. However, the results of our study are not in accordance with the above-mentioned claim. Although MB in a dose of 250 mg did not exert oxidative damage and improved lactase activity, but hematinic activity was found to be less than that of ferrous sulfate. Perhaps treatment with low dose MB for a longer period of time may show its hematinic potential.

Nevertheless, the mechanism by which MB exerts less oxidative stress needs to be explored. It could be either by strengthening the antioxidant defense system or by reducing the free radical generation. These effects were not evaluated in the present study. This could be a limitation of our study. As safety and efficacy of MB have been established in animal model of IDA, a way forward would be to conduct clinical studies in IDA patients which will ascertain the therapeutic effects of this drug in the clinical practice and shed the light on their tolerability so that in future MB can be recommended as an alternative in IDA patients who are intolerant to oral ferrous sulfate therapy.


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