Comparative Anti-dirrhoeal and Antimicrobial Activities of Methanol Extract of Leea indica (Burm. f.) Merr. and Leea macrophylla Roxb. Ex. Hornem (Fam. Vitaceae) and Four Bangladeshi Market Preparations

Introduction

Leea indica (Burm.f.) Merr. (Family: Leeaceae), an evergreen perennial shrub with stout, soft wooded, glabrous stems native to tropical Asia, Australasia, Pacific and grown habitually in Bangladesh, India, China, Bhutan and Malaysia [1]. Traditionally, the whole plant is used to mitigate headache, body pain, and skin complaints [2]. Specifically, leaves and roots of L. indica are traditionally used for the management of cancer, diabetes, diarrhea, dysentery, spasm and skin diseases [3]. Combined root paste of this plant along with the root of Oreocnide integrifolia and Cissus repens in bubo and boils are prescribed by Marma tribes of Chittagong Hill Tracts, Bangladesh [4]. Previous pharmacological studies reported that it possesses antioxidant [3,5,6], analgesic [7], CNS depression [8], phosphodiesterase and nitric oxide synthase inhibitory [9], hepatoprotective [2], cytotoxic [3,4] anticancer [1,10] activities.

Leea macrophylla (Roxb.) is locally identified as Hathikana or Hatikana. It is belonging to Leeaceae family, a herb or herbaceous shrub with a very big sized leaf like an elephant ear [11]. The plant is indigenous to North-Eastern India; though, it is distributed to the relatively hotter parts of India, central and eastern Nepal, Bhutan, China, Myanmar, Thailand, Cambodia, and Laos [12]. In many areas of Bangladesh such as Rajshahi, Jessore, And Natore are noteworthy for the habitat of L. macrophylla [13]. Regarding the ethnobotanical survey, this plant shows some important therapeutic indications in cancer, dysentery, body-ache, and sexual disability [14]. Traditionally, it has some other uses for tonsillitis, tetanus, nephrolithiasis, rheumatism, arthritis, snake bites, sore, pain, and blood effusion [15,16]. This plant is widely used by the ayurvedic physicians in the preparation of seasonal tonic modaka preparation [11]. The juice from the leaf is recognized as local anti-inflammatory agent and used in boils, arthritis, gout, and rheumatism [17]. Seed extract of this plant showed the presence of carbohydrate, protein, glycosides, phenolics, and saponin. The extracts also displayed antifungal activities against Candida albicans except the extract n-hexane [18]. Several literatures revealed anti-inflammatory and analgesic [19], antinociceptive and cytotoxic [20], and urolithiatic [21] effects of leaf extract of this plant. Root extract shows antioxidant, and antibacterial activites [22].

Therefore taking these into consideration, the overall aim of the present study is to detect different chemical groups of the methanol leaf extract of the plants (L. indica and L. macrophylla) and to compare the anti-diarrheal, and antimicrobial activity with four established market products.

Materials and Methods

Plant material

For the investigation, the fresh leaves of Leea indica and Leea macrophylla were collected from the hill of Khamarpara, Rangamati, Chittagong, Bangladesh. The extraneous, undesired substances from the plant material were removed at two stages of processing of it. At first the rotten leaves, stems etc. were removed by hands immediately after collection of the leaves. Again, the soil was removed by sieving through a net aided by a flow of air from an electric fan before the plant materials are dried.

The plants L. indica and L. macrophylla were selected based on its medicinal uses. The traditional practitioners called as “Kabiraj” and the tribal people of hill tracts were the main source of reliable information about the traditional uses of this plant. Taxonomical identification of this plant was made by the experts of Bangladesh Forest Research Institute Herbarium (BFRIH), Chittagong.

Extraction of plant material

The collected leaves were separated from undesirable materials (plant parts or dust) or plants and were shed-dried (35-50ºC). The leaves were ground into coarse powder with the help of a grinder. The powder were stored in airtight containers and kept in a cool, dark and dry place until extraction commenced.

The extracts were weighed separately with the help of electronic and digital balance.

Then the yield was determined by using the following formula:

Yield=[wt of crude extract (g)/wt of initial powder taken (g)] × 100

The amount of crude extracts of L. indica, L. macrophylla from hot extraction was 10 g respectively.

Yield=[10 g/150 g] × 100=6.67%

Animals

Young Swiss-albino mice, average weight of 18-25 g of either sex were employed in the experiment. The mice were collected from the Animal Research Branch of the Bangladesh Council of Scientific and Industrial Research (BCSIR), Chittagong, Bangladesh. The mice were kept separately in wooded cages having dimension of 15 × 10 × 8 inch. Soft wood shavings were placed in the cages for housing of the mice. The mice were housed in a well-ventilated air and lightened house.

Phytochemical investigation

The extracts were subjected to preliminary phytochemical investigation. Small quantity of freshly prepared methanol extract of L. indica, L. macrophylla were subjected to preliminary quantitative phytochemical investigation for detection of phytochemicals such as alkaloids, glycosides, steroids, tannins, flavonoids, saponins, reducing sugars, gums & mucilage using the standard methods [23-29].

In vitro anti-dirrhoeal activity

Preparation of samples for the test, standard and control groups

In the present work, 500 mg/kg and 250 mg/kg doses were selected for plant species and for this, 500 and 250 mg of plant extracts were dissolved respectively in small amount of Tween-80 then the final volumes were adjusted by distilled water.

For reference standard group, 3 mg of Loperamide was dissolved in 10 ml distilled water and so for the four market preparations. For control group small amount of (1%) Tween-80 was mixed with distilled water and final volume was adjusted to make 10 ml.

Experimental design

The method, described by Shoba and Thomas [30] was followed for this study. The animals were all screened initially by giving 0.4 ml of castor oil and only those showing diarrhea were selected for the final experiment. The animals were then, divided into control, positive control and two test groups containing three mice in each group. Control group received vehicle (1% Tween-80 in water) at a dose of 10 ml/kg body weight orally. The positive control group received Loperamide at the dose of 3 mg/kg orally and same dose were taken for the four market preparation; test groups received the methanol extract of L. indica and L. macrophylla respectively; at the dose of 500 mg/kg and 250 mg/kg body weight orally. Each animal was placed in an individual cage, the floor of which was lined with blotting paper. The floor lining was changed every hour. Diarrhea was induced by oral administration of 0.4 ml castor oil to each mouse, 30 mins after the above treatments. During the observation period (4 hrs), the total latency periods (first diarrheal stool after the administration of castor oil) and the number of diarrheic feces excreted by the animals were recorded. A numerical score based on stool consistency was assigned (normal stool=1 and watery stool=2).

Determination of in vitro anti-diarrhoeal activity

Anti-diarrheal episode was determined by evaluating the latency period and diarrhoeal frequency by counting the fecal time and number of the test groups in comparison to the control and standard drug Loperamide groups.

In vitro antimicrobial activity

Antimicrobial screening

The antibacterial and antifungal activities of the crude extracts were evaluated by the disc diffusion method Aboaba and Efuwape, [31] against 4 Gram positive and 6 Gram negative pathogenic bacteria and 7 fungi using ciprofloxacin and fluconazole as standards. All the microbial species were collected from the Microbiology Lab of Department of Pharmacy, Southern University Bangladesh, and Chittagong, Bangladesh. The antimicrobial activity of the test agents was expressed by measuring the diameter of zone of inhibition expressed in mm. The experiments were carried out in triplicate.

Minimum Inhibitory Concentration (MIC)

The minimum inhibitory concentration (MIC) of all the extract was determined by the serial dilution technique [32] in nutrient broth medium, containing graded concentration of the plant extracts and inoculated test organisms.

Conclusion

The present pharmacological study and comparative evaluation of methanol extracts of L. indica and L. macrophylla indicates that the plant contains effective bioactive compounds that can be used for the treatment of diarrheal and microbial diseases and can be a potential source of biologically important drug candidates. However, further research is necessary to find the different lead compounds, isolate them in pure form and determine their full spectrum of efficacy, their safety and feasibility of use of them on human subjects.

Competing Interests

The authors declare that they have no competing interests.

Acknowledgement

The authors greatly acknowledge to respective authority of the Department of Pharmacy, Southern University Bangladesh (SUB), for providing valuable laboratory facilities.

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